α-3 sialyltransferase

ABSTRACT

There are provided a novel α2→3 sialyltransferase expressed by a cloned gene from animal cells, a cDNA encoding the α2→3 sialyltransferase, a method for detecting or suppressing the expression of an α2→3 sialyltransferase by use of said cDNA, a recombinant vector containing said cDNA, a cell containing said vector, and their production processes.

This application is a division of application Ser. No. 07/991,587, filedDec. 16, 1992, now U.S. Pat. No. 5,384,249.

FIELD OF THE INVENTION

The present invention relates to a novel α2→3 sialyltransferase, DNAencoding the α2→3 sialyltransferase, recombinant vector in which the DNAis incorporated and a cell containing the recombinant vector as well asa process for producing them. Further, the present invention relates toa process for producing a sugar chain using the α2→3 sialyltransferaseand a process for producing a sugar chain by expressing the α2→3sialyltransferase in a transformed cell. Further, the present inventionrelates to a method for detecting or inhibiting the expression of α2→3sialyltransferase using cDNA encoding the α2→3 sialyltransferase of thepresent invention. The α2→3 sialyltransferase of the present inventionis useful for producing a sugar chain or modification thereof havinguseful physiological activity and improving a sugar chain which isattached to a useful physiologically active protein.

BACKGROUND OF THE INVENTION

While proteins produced by a procaryote such as Escherichia coli and thelike have no sugar chain, the proteins and lipids produced by aneucaryote such as yeast, fungi, plant cells, animal cells and the likehave an attached sugar chain in many cases.

As a sugar chain in animal cells, N-linked sugar chain (also called asN-glycan) which binds to asparagine (Asn) residue in proteins, andO-linked sugar chain (also called as O-glycan) which binds to serine(Ser) or threonine (Thr) residue are known to be added to glycoproteins.There has been recently revealed that a certain lipids containing asugar chain are bound covalently to a number of proteins and theproteins are attached to the cell membrane via those lipids. Thoselipids containing a sugar chain are called as glycosylphosphatidylinositol anchor.

The other example of a sugar chain in animal cells is glycosaminoglycan.A compound wherein a protein and a glycosaminoglycan are covalentlybound is called as proteoglycan. Although glycosaminoglycan which is acomponent of sugar chain of proteoglycan has similar structure to thatof O-glycan which is glycoprotein sugar chain,glycosaminoglycan-has-chemical properties different from those ofO-glycan. Glycosaminoglycan has the characteristic structure composed ofdisaccharide unit repeats containing glucosamine or galactosamine anduronic acid (except that keratan sulfate has no uronic acid), whereinthe sulfate groups are covalently bound thereto (except that hyaluronicacid has no sulfate groups).

Furthermore, as a sugar chain in animal cells, there is a sugar chaincontained in glycolipid. As glycolipid in animal cells, there are knownsphingoglycolipid in which sugar, long chain fatty acid and sphingosinewhich is long chain base are covalently bound, and glyceroglycolipid inwhich sugar chain is covalently bound to glycerol.

Recently, elucidation on the function of a sugar chain has been rapidlyadvanced together with advance in molecular biology and cell biology,and a variety of functions of a sugar chain have been revealed. Firstly,a sugar chain play an important role on clearance of glycoprotein inblood. Erythropoietin obtained by transferring a gene in Escherichiacoli manifests in vitro its activity, but is known to be rapidly in vivoclearanced [Dordal et al.: Endocrinology, 116, 2293 (1985) and Browne etal.: Cold Spr. Harb. Symp. Quant. Biol., 51, 693, 1986]. Humangranulocyte-macrophage colony stimulating factor (hGM-CSF) has naturallytwo N-linked sugar chains, but it is known that, as the-number of sugarchains is decreased, the clearance rate in rat plasma is raisedproportionally thereto [Donahue et al.: Cold Spr. Harb. Symp. Quant.Biol., 51, 685 (1986)]. The clearance rate and clearanced places varydepending upon the structure of a sugar chain. It is known that, whilehGM-CSF to which sialic acid is added is clearanced in kidney, hGM-CSFfrom which sialic acid is removed is raised in the clearance rate and isclearanced in liver. Additionally, the clearance rates in rat plasma andrat perfusion liquid were studied with respect α1-acid glycoproteinshaving different sugar chain which were biosynthesized by rat liverprimary culture in the presence of various N-linked sugar chainbiosynthesis inhibitors. In both cases, the clearance rates were slowerin descending order of high mannose type, sugar chain deficient type,hybrid type and complex type (natural type). It is also known that theclearance in blood of tissue-type plasminogen activator (t-PA) used as afibrinolytic agent is significantly influenced by the structure of asugar chain.

It is also known that a sugar chain endows a protein with the proteaseresistance. For example, when formation of sugar chain of fibronectin isinhibited by zunicamycin, the resulting sugar chain deficientfibronectin is promoted in the intracellular degrading rate. It is alsoknown that addition of a sugar chain increases the heat stability andanti-freezing properties. It is also known that a sugar chain makes acontribution to increase the solubility of protein.

A sugar chain also makes a contribution to holding the correct stericstructure of proteins. It is known that, although the removal of twoN-linked sugar chains naturally present in membrane-bound glycoproteinof vesicular stomatitis virus inhibits the transport of proteins to cellsurface, new addition of a sugar chain to said proteins recovers thetransport. In this case, it has been revealed that the removal of asugar chain induces the association between protein molecules viadisulfide bond and, as the result, the transport of proteins isinhibited. Since the correct steric structure is retained due toinhibition of this association by new addition of sugar chain, thetransport of proteins becomes possible. It is shown that the position towhich new sugar chain is added is considerably flexible. To thecontrary, it has been revealed that the transport of proteins havingnatural sugar chains is completely inhibited, in some cases, dependingupon the introduction site of the additional sugar chain.

There is known the case where a sugar chain masks antigen site onpolypeptide. From the experiments using polyclonal antibody ormonoclonal antibody reacting with a particular region on polypeptide inhGM-CSF, prolactin, interferon-γ, Rauscher leukemia virus gp70 andinfluenza hemagglutinin, it is considered that a sugar chain of theabove proteins inhibits a reaction with antibody. There is also knownthe case where a sugar chain itself has direct relationship withmanifestation of the activity of glycoprotein. For example, a sugarchain is considered to participate in manifestation of the activity ofglycoprotein hormones such as luteinizing hormone, follicule stimulatinghormone, chorionic gonadotropin and the like.

In addition, EP-A 0370205 discloses that granulocyte colony-stimulatingfactor (G-CSF), pro-urokinase (pro-UK) and the like can be improved inthe properties by artificially and intentionally introducing a sugarchain into the proteins using recombinant DNA techniques.

Furthermore, as an important function of a sugar chain, there isparticipation in recognition phenomena between cells, between proteinsor between cells and proteins. For example, it is known that the placewhere a sugar chain is clearanced in the living body is differentdepending upon the difference in the structure of a sugar chain. Inaddition, it has been found that a ligand of ELAM-1 which isinflammatory response-specifically expressed on blood vessel endothelialcell and promotes adhesion to neutrophil is a sugar chain called asSialyl-Le^(X) [NeuAc α2→3Gal β1→4 (Fuc α1→3)GlcNAc:NeuAc, sialic acid;Gal, galactose; Fuc, fucose; GlcNAc, N-acetylglucosamine]. As theresult, there has risen the possibility that a sugar chain itselfor-modification thereof is used in pharmaceuticals and the like[Phillips et al.: Science 250, 1130 (1990), Goelz et al.: Trends inGlycoscience and Glycotechnology 4, 14 (1992)]. Further, it is suggestedthat L-selectin which is expressed in a part of T lymphocytes andneutrophil and GMP-140 (also called as P-selectin) which is expressed inthe membrane surface of platelet and blood vessel endothelial cell byinflammatory stimulation participate in inflammatory response as same asELAM-1, and ligands thereof are also sugar chains similar toSialyl-Le^(X) sugar chain which is a ligand of ELAM-1 [Rosen et al.:Trends in Glycoscience and Glycotechnology 4, 1 (1992), Larsen et al.:Trends in Glycoscience and Glycotechnology) 4, 25 (1992), Aruffo et al.:Trends in Glycoscience and Glycotechnology 4, 146 (1992)].

Also in metastasis of cancer as in the inflammatory response, it issuggested that ELAM-1 and GMP-140 promote metastasis of cancer bycausing adhesion of cancer cells to inner wall of blood vessel oraggregation between cancer cells and platelets [Goelz et al.: Trends inGlycoscience and Glycotechnology) 4, 14 (1992), Larsen et al.: Trends inGlycoscience and Glycotechnology) 4, 25 (1992)]. These suggestions areconsistent with the findings that expression of Sialyl-Le^(X) sugarchain is high in cancer cells having high metastasis ability [Irimura etal.: Experimental Medicine 6, 33 (1988)].

From these findings, it is expected that Sialyl-Le^(X) sugar chain orderivatives thereof manifest the excellent anti-inflammatory effects andanti-metastatic effects by binding to ELAM-1, L-selectin or GMP-140.

Additionally, in view of the mechanism of the above-describedinflammatory response and metastasis of cancer, inflammatory responsecould be inhibited and metastasis of cancer could be prevented byinhibiting the expression of glycosyltransferase which controlssynthesis of ligand sugar chain recognized by ELAM-1, L-selectin orGMP-140. Antisense RNA/antisense DNA techniques [Tokuhisa: Bioscienceand Industry 50, 322 (1992), Murakami: Chemistry 46, 681 (1991)] orTriple helix techniques [Chubb and Hogan: Trends in Biotechnology 10,132 (1992)] are useful for inhibiting the expression of some particulargenes. Since information on those gene or nucleotide sequence of thosegene is necessary in order to inhibit the expression of desiredglycosyltransferase using this antisense RNA/DNA techniques, the cloningof a gene of desired glycosyltransferase and analysis of the informationon the nucleotide sequence are important.

Further, diagnosis of malignancy in inflammatory diseases or cancer canbe also performed by investigating the expression of the particularglycosyltransferase in inflammatory lymphocyte and cancer cells. Forinvestigating the expression of desired glycosyltransferase, Northernhybridization method which uses as a probe the relevant gene labelledwith radioactivity and the like [Sambrook, Fritsch, Maniatis, MolecularCloning, A laboratory manual, 2nd edition, Cold Spring Harbor LaboratoryPress, 1989] and Polymerase Chain Reaction method (abbreviated as PCRmethod hereinafter) [Innis et al.: PCR Protocols, Academic Press, 1990]are useful. For applying these methods, the information on desiredglycosyltransferase gene or nucleotide sequence thereof is necessary.Also from this respect, the cloning of desired glycosyltransferase geneand analysis of information of that nucleotide sequence are important.

As described above, alteration in the structure of glycoprotein and massproduction of the particular sugar chain or modification thereof areindustrially extremely important themes.

The means by which the structure of a sugar chain is altered haverecently advanced remarkably. In particular, the structure of a sugarchain can be altered by a high specific enzyme (exoglycosidase) whichsuccessively dissociates a sugar chain or glycopeptidase and endo-typeglycosidase which cleavages a bonding between peptide chain and sugarchain without changing both peptide chain and sugar chain. As theresult, the biological role of a sugar chain can be studied in detail.Further, endoglycoceramidase which cleavages between a sugar chain ofglycolipid and ceramide has been recently found [Ito and Yamagata: J.Biol. Chem. 261, 14278 (1986)]. This finding has not only facilitatedthe preparation of a sugar chain of glycolipid but also advanced thestudy on the function of cell surface glycolipid. In addition, newaddition of sugar chain has been possible by using glycosyltransferase.For example, sialic acid can be newly added to the end of sugar chain byusing sialyltransferase [Sabesan and Paulson: J. Am. Chem. Soc. 108,2068 (1986)]. A sugar chain to be added can be varied by using othervarious glycosyltransferases or glycosidase inhibitors [Alan et al.:Annu. Rev. Biochem. 56, 497 (1097)]. However, mass production ofglycosyltransferase used in synthesis of a sugar chain is extremelydifficult. For that reason, it is desired that glycosyltransferase isproduced in a large amount by cloning glycosyltransferase usingrecombinant DNA techniques and effectively expressingglycosyltransferase in a host cell.

As a method for cloning glycosyltransferase, there are known a method bypurifying a protein, produing an antibody reacting with it andperforming immunoscreening using the antibody [Weinstein et al.: J.Biol. Chem. 262, 17735 (1987)], and a method by purifying a protein,determining amino acid sequence thereof, producing synthetic DNA whichcorresponds thereto and performing hybridization using the DNA as aprobe [Narimatsu et al.: Proc. Natl. Acad. Sci., USA, 83, 4720 (1986)].A method is also known where hybridization is performed using clonedglycosyltransferase gene as a probe and thereby glycosyltransferase genehaving homology with the glycosyltransferase is cloned [John. B. Lowe etal.: J. Biol. Chem. 266, 17467 (1991)]. In addition, there is known acloning method by direct expression cloning using panning method asscreening method, in which antibody or lectin reacting with a sugarchain is employed [John. B. Lowe et al.: Proc. Natl. Acad. Sci.,USA, 86,8227 (1989), John. B. Lowe et al.: Genes Develop., 4, 1288 (1990)].

There is no case where glycosyltransferase can be cloned usinglectin-resistance as an index. From the studies on variouslectin-resistant mutants of CHO cell, it has been revealed that thereare cases where new glycosyltransferase is expressed, where the activityof a certain glycosyltransferase disappears, and where synthesis ofsugar nucleotide or its transfer to Golgi body is inhibited [PamelaStanley et al.: Methods in Enzymology, 96, 157]. Therefore, it isconsidered that cloning of glycosyltransferase can be performed usinglectin-resistance as index by introducing a gene derived from a cellexpressing glycosyltransferase to be cloned into CHO cell orlectin-resistant mutants of CHO cell [Ravindra Kumar et al.: Mol. Cell.Biol., 9, 5713 (1989)]. James Ripka et al. have tried to cloneN-acetylglucosaminyltransferase I by introducing human genomic DNAderived from A431 cell into lectin-resistant mutants of CHO cell (Lecl)using resistance to lectin concanavalin A as an index. However, theycould not clone glycosyltransferase by the screening method usinglectin-resistance as an index [James Ripka et al.: Biochem. Biophys.Res. Commun., 159, 554 (1989)]. Heffernan et al. have cloned mousesialic acid hydroxylase using resistance to lectin WGA (wheat germagglutinin) as an index by introducing cDNA library into CHO cell[Michael Heffernan et al.: Nucleic Acids Res., 19, 85 (1991)] which wasmade to produce large T antigen of polyoma [Michael Heffernan et al.:Glycoconjugate J., 8, 154 (1991)]. However, there is no report in whichglycosyltransferase could be cloned in a screening system using thelectin-resistance as an index. In addition, with respect to hosts,Stanley, Ripka, Heffernan et al. all used CHO cell or lectin-resistantmutants of CHO cell as a host.

With respect to sialyltransferase, a cDNA encoding an enzyme havingβgalactoside α2→6 sialyltransferase activity has been isolated andnucleotide sequence thereof has been revealed [Weinstein et al.: J.Biol. Chem., 262, 17735 (1987)]. With respect to an enzyme havingβgalactoside α2→3 sialyltransferase activity, Gillespie et al. havereported cloning of a gene encoding an enzyme which adds sialic acid togalactose in O-linked sugar chain of glycoprotein (sugar chain which isadded to serine or threonine residue), but base sequence thereof has notbeen revealed [Gillespie et al.: Glycoconjugate J., 7, 469 (1990)]. Inaddition, Weinstein et al. have been reported a method for purifying anenzyme having βgalactoside α2→3 sialyltransferase activity from ratliver [Weistein et al.: J. Biol. Chem., 257, 13835 (1982)]. However,desired enzyme can be obtained in an extremely small amount. Hitherto,there have been no reports in which sialic acid is added in α2→3 linkageto desired position on sugar chain of glycoprotein, glycolipid,oligosaccharide and the like using recombinant DNA techniques.

OBJECTS OF THE INVENTION

The main object of the present invention is to provide novel α2→3sialyltransferase which effectively alters a sugar chain of protein andproduces a particular sugar chain, cDNA encoding said α2→3sialyltransferase and a vector containing said cDNA.

These objects as well as other objects and advantages of the presentinvention will become apparent to those skilled in the art from thefollowing description with reference to the accompanying drawings.

SUMMARY OF THE INVENTION

The present inventors constructed cDNA library by incorporating cDNAwhich was synthesized using mRNA extracted from animal cells as atemplate into expression cloning vector, introduced said cDNA libraryinto a cell, the resultant cell was cultured in the presence of lectinhaving the activity which inhibits growth of said cell, and growingcells were isolated to obtain a cloned gene which was introduced in ahost cell to express. As the result, we found that novel α2→3sialyltransferase was expressed, resulting in completion of the presentinvention.

According to the present invention, there is provided novel α2→3sialyltransferase having amino acid sequence shown by SEQUENCEIDENTIFIER NUMBER (Seq. ID:) 2 or 7, cDNA encoding said α2→3sialyltransferase, and a recombinant vector containing said DNA. α2→3Sialyltransferase of the present invention is glycosyltransferase havingthe βgalactoside α2→3 sialyltransferase activity, and has the activitywhich adds sialic acid to end of substrate sugar chain in α2→3 linkage.

BRIEF EXPLANATION OF THE DRAWING

FIG. 1 is a flow sheet showing construction of plasmid pAGEL106.

FIG. 2 is a flow sheet showing construction of plasmid pASLB3-3-1.

FIG. 3 is a flow sheet showing construction of plasmid pASLB3-3.

FIG. 4 is a flow sheet showing construction of plasmid pSALBE3-3.

FIG. 5 is a flow sheet showing construction of plasmid pASLBC.

FIG. 6 is a flow sheet showing construction of plasmid pASLBEC.

FIG. 7 is a flow sheet showing construction of plasmid pASLBEC2.

FIG. 8 is a flow sheet showing construction of plasmid pAMoEC2.

FIG. 9 is a flow sheet showing construction of plasmid pAMoEC3.

FIG. 10 is a flow sheet showing construction of plasmid pAMoERC3.

FIG. 11 is a flow sheet showing construction of plasmid pAGE207.

FIG. 12 is a flow sheet showing construction of plasmid pAGE207ScN.

FIG. 13 is a flow sheet showing construction of plasmid pAMoC3Sc.

FIG. 14 is a flow sheet showing construction of plasmid pAMoERC3Sc.

FIG. 15 is s flow sheet showing construction of cDNA library.

FIG. 16 is a flow sheet showing construction of plasmid pUCl19-LEC.

FIG. 17 is a view showing the results of the sialyltransferase activitymeasured by HPLC. Pannels a. and b. show HPLC pattern on KJM-1 strain inwhich pAMoERL is introduced and Pannels c. and d. show HPLC pattern onKJM-1 strain in which vector pAMoERC3Sc is introduced, respectively.Pannels a. and c. show HPLC pattern where an assay solution containingCMP-sialic acid as sugar donor is used and Pannels b. and d. show HPLCpattern where an assay solution not containing CMP-sialic acid is used,respectively.

FIG. 18 is a view showing the results of analysis by HPLC after peak 1and peak 2 are treated with sialidase. Pannels a. and c. show HPLCpattern on peak 1 and peak 2 without sialidase treatment, respectively.Pannels b. and d. show HPLC pattern on peak 1 and peak 2 after sialidasetreatment, respectively. Peak 3 shows the product obtained by treatingpeak 1 and peak 2 with sialidase.

FIG. 19 is a view showing the results of analysis by a flow cell sorterFCS-1 (manufactured by Nihonbunko) after indirect fluorescent antibodystaining. Pannel a shows the results of indirect fluorescent antibodystaining using KM93 or serum of normal mouse on KJM-1 in whichpAMoERC3Sc is introduced. Dotted line shows the pattern when normalmouse serum is used, and solid line shows the pattern when KM93 is used.Pannel b shows the results of indirect fluorescent antibody stainingusing KM93 on KJM-1 strain in which pAMoERL is introduced. Dotted lineis the same one shown in Pannel a., and solid line shows the patternwhen KM93 is used.

FIG. 20 is a flow sheet showing construction of plasmid pAGE147.

FIG. 21 is a flow sheets showing construction of plasmid pAGE247.

FIG. 22 is a flow sheet showing construction of plasmid pAMN6hyg.

FIG. 23 is a flow sheet showing construction of plasmid pAMoERSA.

FIG. 24 is a flow sheet showing construction of plasmid pAMoPRC3Sc.

FIG. 25 is a flow sheet showing construction of plasmid pAMoPRSA.

FIG. 26 is a flow sheet showing construction of plasmid pAMoPRSAL-35F.

FIG. 27 is a flow sheet showing construction of plasmid pUCl19-WM17.

FIG. 28 is a view showing the results of analysis by EPICS Elite FlowCytometer (manufactured by COULTER) after indirect fluorescent antibodystaining. As a control, the results of indirect fluorescent antibodystaining using normal mouse serum on KJM-1 strain in which pAMoPRC3Sc(control plasmid) is introduced are also shown. In addition, the resultsof indirect fluorescent antibody staining using KM93 on KJM-1 strain inwhich pAMoPRC3Sc (control plasmid) or pAMoPRWM17 (α2→3 sialyltransferaseexpression plasmid) is introduced are also shown as pAMoPRC3Sc orpAMoPRWM17, rescpectively.

FIG. 29 is a flow sheet showing construction of plasmid pAMoPRSAW17-31F.

The abbreviations used herein have the following meanings.

dhfr: dihydrofolate reductase gene

hG-CSF: human granulocyte colony stimulating factor gene

bp: base pairs

kb: kilobase pairs

G418/Km: G418 and kanamycin resistance gene derived from transposon 5(Tn5)

hyg: hygromycin resistance gene

Ap: ampicillin resistance gene derived from pBR322

Tc: tetracycline resistance gene derived from pBR322

P1: P1 promoter derived from pBR322

Pkt: Herpes simplex virus (HSV) thymidine kinase (tk) gene promoter

Sp. βG: rabbit βglobin gene splicing signal

A. βG: rabbit βglobin gene poly A addition signal

A. SE: simian virus 40 (SV40) early gene poly A addition signal

Atk: Herpes simplex virus (HSV) thymidine kinase (tk) gene poly Aaddition signal

Pse: simian virus 40 (SV40) early gene promoter

Pmo: Moloney murine leukemia virus long terminal repeat (LTR) promoter

HTLV-1: human T-cell leukemia virus type-1 (HTLV-1) gene

EBNA-1: Epstein-Barr virus EBNA-1 gene

oriP: Epstein-Barr virus replication gene

ori: pUCl19 replication gene

lac'Z: a part of Escherichia coli βgalctosidase gene

IG: intergenic region of M13 phage DNA

DETAILED DESCRIPTION OF THE INVENTION

cDNA encoding α2→3 sialyltransferase of the present invention include(a) DNA having nucleotide sequence shown in Seq. ID: 1 or 6, (b) DNAhaving nucleotide sequence different from that shown in Seq. ID: 1 or 6due to presence of a plurality of genetic codes relative to one aminoacid, or due to natural mutation which occurs in individual animalincluding human being, (c) DNA in which mutations such as substitution,deletion, insertion and the like are introduced in the DNA defined by(a) or (b) without losing the activity of α2→3 sialyltransferaseactivity of the present invention, for example, DNA having such homologywith α2→3 sialyltransferase encoded by DNA defined by (a) or (b) as canbe isolated by hybridization method. α2→3 Sialylytransferase of thepresent invention includes all of α2→3 sialyltransferases encoded by DNAdefined in the above (a), (b) or (c).

A process for producing cDNA which encodes α2→3 sialyltransferase of thepresent invention is explained below by exemplifying a process forproducing cDNA defined in the above (a).

cDNA library is constructed by incorporating cDNA synthesized using mRNAextracted from animal cells as a template into expression cloningvector. This cDNA library is introduced in animal or insect cells, andthe cells are cultured in the presence of lectin which inhibits thegrowth of the cells. A cell transfected with cDNA encoding appropriateglycosyltransferases which change the structure of sugar chainrecognized by lectin grows in the presence of lectin. This cell isisolated, and cDNA encoding desired α2→3 sialyltransferase is obtainedfrom the cell.

As an animal cell used in the above method, any animal cell can be usedso long as cDNA encoding α2→3 sialyltransferase of the present inventioncan be expressed in the animal cell. For example, human histiocyticleukemia cell line TYH [Haranaka et al.: Int. J. Cancer, 36, 313(1985)], human melanoma cell line WM266-4 (ATCC CRL1676) and the likeare used. As a vector in which cDNA synthesized using mRNA extractedfrom these cells as a template is incorporated, any vectors can be usedin which said cDNA can be incorporated and expressed. For example,pAMoERC3Sc and the like are used. As an animal or insect cell in whichcDNA library constructed using said vector is introduced, any cells canbe used in which said cDNA library can be introduced and expressed. Forexample, human Namalwa cell [Hosoi et al.: Cytotechnology, 1, 151(1988)] and the like can be used. As lectin to be used in the presentinvention, any lectins can be used which can inhibit the growth of cellin which cDNA is introduced. For example, Ricinus communis 120 lectinand the like are used. After resistance of host cell to lectin isdetermined, the lectin is used in such the concentration as can inhibitthe growth of host cell. Plasmid having cDNA encoding α2→3sialyltransferase of the present invention or DNA fragment containingthe cDNA part is recovered from cells which grow in the presence oflectin by the known method, for example, Hirt method [Robert F.Margolskee et al.: Mol. Cell. Biol., 8, 2837 (1988)]. As a plasmidhaving cDNA encoding the enzyme of the present invention, there are, forexample, pUCl19-LEC, pUCl19-WM17 and the like. Escherichia coliHB101/pUCl19-LEC containing pUCl19-LEC and Escherichia coliHB101/pUCl19-WM17 have been deposited with Fermentation ResearchInstitute, Agency of Industrial Science and Technology, 1-3, Higashi 1chome Tsukuba-shi Ibaraki-ken 305, Japan on Oct. 29, 1991 and on Sep.22, 1992 under the Budapest Treaty, and have been assigned the accessionnumber FERM BP-3625 and FERM BP-4013, respectively.

DNA defined by the above (b) and (c) can be prepared by well knownrecombinant DNA techniques such as hybridization method, and a methodintroducing mutations in DNA and the like, based on cDNA encoding α2→3sialyltransferase obtained by the above process. Alternatively, cDNAencoding α2→3 sialyltransferase of the present invention can be preparedusing chemosynthetic method.

DNA encoding α2→3 sialyltransferase of the presernt invention obtainedin the above process is inserted downstream of appropriate promoter toconstruct recombinant vector which is introduced in a host cell, and theresulting cell is cultured to obtain α2→3 sialyltransferase of thepresent invention. As a host cell, any host cells can be used to whichrecombinant DNA techniques have been applied such as procaryotic cells,animal cells, yeasts, fungi, insect cells and the like. For example,there are Escherichia coli cell as procaryotic cell, CHO cell which isChinese hamster ovary cell, COS cell which is monkey cell, Namalwa cellwhich is human cell and the like as animal cell. In particular, directexpression system using Namalwa cell as a host cell is suitably used dueto such the advantages that efficiency which introduces cDNA library inNamalwa cell as a host cell is extremely high, the introduced plasmid(cDNA library) can exist extrachromosomally, and plasmid is easilyrecovered from the resultant lectin resistant strain.

As a vector for introducing DNA encoding the present α2→3sialyltransferase therein, there can be used any vectors in which DNAencoding the α2→3 sialyltransferase can be incorporated and which can beexpressed in a host cell. For example, there are pAGE107 [JP-A 3-22979,Miyaji et al.: Cytotechnology, 3, 133 (1990)], pAS3-3 [EP-A 0370205],pAMoERC3Sc, CDM8 [Brian Seed et al.: Nature, 329, 840 (1987)] and thelike. For expressing the cDNA encoding the enzyme of the presentinvention in Escherichia coli, it is preferred that there is used aplasmid wherein foreign DNA can be inserted downstream of promoterhaving the strong transcription activity such as trp promoter and thelike and the distance between Shine-Dalgarno sequence (abbreviated as SDsequence hereinafter) and initiation codon is appropriately adjusted(for example, said distance is 6 to 18 bases). More particularly, thereare pKYP10 [EP-A 0083069], pLSA1 [Miyaji et. al: Agric. Biol. Chem., 53,277 (1989)], pGEL1 [Sekine at. al, Proc. Natl. Acad. Sci., USA, 82, 4306(1985)] and the like.

As general procedures of recombinant DNA techniques used in the presentinvention, there can be used those described in EP-A 0370205 or thosedescribed by Sambrook, Fritsch, Maniatis et al. [Molecular Cloning, Alaboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press,1989]. Isolation of mRNA and synthesis of cDNA library can be effectedusing the above-described methods and many kits on the market. Forintroducing DNA in an animal cell, there can be used any currently knownmethods. For example, electroporation method [Miyaji et al.:Cytotechnology, 3, 133 (1990)], calcium phosphate method [EP-A 0370205],lipofection method [Philip L. Felgner et al.: Proc. Natl. Acad. Sci.,USA, 84, 7413 (1987)] and the like can be used. Obtaining of transfectedcells and culturing them can be performed according to the methoddescribed in EP-A 0370205 or JP-A 2-257891.

As a process for producing cloned α2→3 sialyltransferase, there are amethod for expressing it in a host cell, a method for secreting it froma host cell, and a method for expressing it on cell surface of a hostcell. The place where expression is effected varies depending upon thekind of host cell to be used and upon the form of glycosyltransferase tobe produced. When glycosyltransferase is produced as it is, using ananimal cell as a host cell, it is generally produced in a host cell oron cell surface of a host cell, and a part of it is extracellularlyexcreted upon cleavage by protease. To secrete glycosyltransferaseintentionally, it is produced in a form where a signal peptide is addedto a part containing an active site of glycosyltransferase usingrecombination DNA techniques according to a method by Paulson et al. [C.Paulson et al.: J. Biol. Chem., 264, 17619 (1989)] and a method by Loweet al [John B. Lowe et al.: Proc. Natl. Acad. Sci., USA, 86, 8227(1989), John B. Lowe et al.: Genes Develop., 4, 1288 (1990)].

Alternatively, the production yield can be increased using geneamplification system using dihydrofolate reductase gene and the likeaccording to a method described in EP-A 0370205.

α2→3 Sialyltransferase of the present invention thus produced can bepurified by a conventional purifying method for glycosyltransferase [J.Evan. Sadler et al.: Methods of Enzymology, 83, 458]. When the enzyme isproduced in Escherichia coli, it can be effectively purified incombination of the above method and the method described in EP-A0272703. Alternatively, purification can be carried out by producing theenzyme of the present invention as a fused protein with other proteinand subjecting it to affinity chromatography using substance having theaffinity with the fused protein. For example, the enzyme of the presentinvention can be produced as a fused protein with protein A and purifiedby affinity chromatography using immunoglobulin G according to themethod by Lowe et al. [John. B. Lowe et al.: Proc. Natl. Acad. Sci.,USA, 86, 8227 (1989), John. B. Lowe et al.: Genes Develop., 4, 1288(1990)]. Alternatively, the enzyme can be purified by affinitychromatography using an antibody reacting with the enzyme itself.

The activity of sialyltransferase is determined by the known method [J.Evan. Sadler et al.: Methods in Enzymology, 83, 458, Naoyuki Taniguti etal.: Methods in Enzymology, 179, 397].

A sugar chain can be synthesized in vitro using α2→3 sialyltransferaseof the present invention. For example, sialic acid can be added in α2→3linkage to non-reducing end-group of lactosamine structure (Galβ1→4GlcNAc structure) contained in glycoprotein, glycolipid oroligosaccharide. The structure of sugar chain at non-reducing terminuscan also be converted to Sialyl-Le^(X) structure by acting α2→3sialyltransferase of the present invention to glycoprotein, glycolipidor oligosaccharide as a substrate. An oligosaccharide havingSialyl-Le^(X) and modification thereof at non-reducing terminus can besynthesized using known α1→3 fucosyltransferase [Lowe et al.: GenesDevelop., 4, 1288 (1990), Goelz et al.: Cell, 63, 1349 (1990)] afteracting α2→3 sialyltransferase of the present invention to anoligosaccharide having lactosamine structure at non-reducing terminus.

DNA encoding α2→3 sialyltransferase of the present invention can be usedto be expressed together with DNAs encoding glycoprotein, glycolipid oroligosaccharide having the useful physiological activity in an animalcell or insect cell producing a sugar chain substrate to the α2→3sialyltransferase, and the produced α2→3 sialyltransferase can be actedon glycoprotein, glycolipid or oligosaccharide to obtain glycoprotein,glycolipid or oligosaccharide having the altered sugar chain structure.

Further, a part of oligosaccharide can be excised from the resultingglycoprotein, glycolipid or oligosaccharide having the altered sugarchain structure by using the known enzymatic or chemical method.

DNA encoding α2→3 sialyltransferase of the present invention can be usednot only in modification of sugar chain of glycoproteins, glycolipids oroligosaccharides and efficient production of the particular sugar chainbut also in therapy for diseases such as inflammation or cancermetastasis using antisense RNA/DNA techniques and diagnosis on thosediseases using Northern hybridization method or PCR method.

Expression of the activity of α2→3 sialyltransferase of the presentinvention can be inhibited using, for example, DNA encoding the α2→3sialyltransferase by antisense RNA/DNA techniques [Tokuhisa: Bioscienceand Industry, 50, 322 (1992), Murakami: Kagaku, 46, 681 (1991), Miller:Biotechnology, 9, 358 (1992), Cohen: Trends in Biotechnology, 10, 87(1992), Agrawal: Trends in Biotechnology, 10, 152 (1992)] or triplehelix technique [Chubb and Hogan: Trends in Biotechnology, 10, 132(1992)]. More particularly, expression of the α2→3 sialyltransferase canbe inhibited by administering in the living body an oligonucleotidewhich is designed and prepared based on nucleotide sequence of a part ofα2→3 sialyltransferase gene, preferably, 10-50 bases sequence in theinitiation region. As nucleotide sequence of synthetic oligonucleotide,there can be used the completely same sequence as that of a part ofantisense strand disclosed herein or a sequence which is modified sothat α2→3 sialyltransferase expression inhibiting activiy is not lost.When triple helix technique is used, nucleotide sequence of syntheticoligonucleotide is designed based on the information about nucleotidesequence of both sense strand and antisense strand.

In addition, expression of cDNA encoding α2→3 sialyltransferase of thepresent invention can be detected by hybridizaiton method or PCR asfollows.

For detecting expression of cDNA encoding α2→3 sialyltransferase of thepresent invention using Northern hybridization method or PCR method,probe DNA or synthetic oligonucleotide is prepared based on cDNAencoding α2→3 sialyltransferase of the present invention or nucleotidesequence thereof. Northern hybridization method and PCR method areperformed according to Molecular Cloning, A laboratory manual, 2ndedition [Cold Spring Harbor Laboratory Press, 1989] and PCR Protocols[Academic Press, 1990].

The following Examples further illustrate the present invention indetail but are not be construed to limit the scope thereof.

In the following Examples, T4 polynucleotide kinase and T4 DNA ligaseused were those manufactured by Takarashuzo K.K.

EXAMPLE 1

1. Construction of direct expression cloning vector pAMoERC3Sc

(1) Construction of pAGEL106 (see FIG. 1)

According to the method described below, plasmid pAGEL106 havingpromoter in which simian virus 40 (SV40) early gene promoter and a partof R region and U5 region of long terminal repeat of human T-cellleukemia virus type-1 (HTLV-1) are fused was constructed. That is, DNAfragment containing a part of R region and U5 region [BanII-Sau3AIfragment (0.27 kb)] was isolated from pATK03 and inserted between BglIsite and BamHI site of pAGE106 via synthetic linker.

pAGE106 (1 μg) obtained by the method described in EP-A 0370205 wasdissolved in a buffer (abbreviated as Y-100 buffer hereinafter)containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride, 100 mMsodium chloride and 6 mM 2-mercaptoethanol, and digested with 10 unitsof BglI (manufactured by Takarashuzo, restriction enzymes were usedthose manufactured by Takarashuzo hereinafter unless others indicated)and 10 units of BamHI at 37° C. for two hours. The reaction solution wassubjected to agarose gel electrophoresis to obtain about 4.9 kb of DNAfragment.

Separately, 1μ of pATK03 [Shimizu at al.: Proc. Natl. Acad. Sci., USA,80, 3618 (1983)] was dissolved in 30 μl of Y-100 buffer, and 10 units ofBanII was added thereto. The mixture was subjected to digestion reactionat 37° C. for two hours, and subjected to agarose gel electrophoresis togive about 0.4 kb of DNA fragment. The resulting DNA fragment wasdissolved in Y-100 buffer (30 μl) and 10 units of Sau3AI was addedthereto. The mixture was subjected to digestion reaction at 37° C. fortwo hours, and subjected to agarose gel elctrophoresis to give about0.27 kb of DNA fragment.

Separately, for linking BglI cleavage site and BanII cleavage site, thefollowing DNA liker was synthesized.

    5'CGGGCT3' (6 mer)

    3'GGAGC5' (5 mer)

The 5 mer and 6 mer single-stranded DNAs of the DNA linker weresynthesized using a DNA synthesizer model 380A (Applied Biosystems).Each 0.2 μg of the synthesized DNAs was dissolved in 40 μl of a buffer(abbreviated as T4 kinase buffer hereinafter) containing 50 mM Tris-HCl(pH 7.5), 10 mM magnesium chloride, 5 mM dithiothreitol (abbreviated asDTT hereinafter), 0.1 nM EDTA and 1 mM adenosine triphosphate(abbreviated as ATP hereinafter) and phosphorylated with 30 units of T4polynucleotide kinase at 7° C. for two hours.

The DNA fragments thus obtained, i.e., 0.2 μg of BglI-BamHI fragment(4.9 kb) derived from pAGE106 and 0.01 μg of BanII-Sau3AI fragment (0.27kb) derived from pATK03 were dissolved in 30 μl of buffer (abbreviatedas T4 ligase buffer hereinafter) containing 66 mM Tris-HCl (pH 7.5), 6.6mM magnesium chloride, 10 mM DTT and 0.1 mM ATP. 0.01 μg of the aboveDNA linker was added to the solution and both the DNA fragments and DNAlinker were ligated together with 175 units of T4 DNA ligase at 12° C.for 16 hours.

Escherichia coli HB101 strain [Bolivar et al.: Gene, 2, 75 (1977)] wastransformed using the above reaction mixture according to the method byCohen et al. [S. N. Cohen et al.: Proc. Natl. Acad. Sci., USA, 69, 2110(1972)] to obtain kanamycin resistant strains. From these transformants,a plasmid was isolated according to the known method [H. C. Birnboim etal.: Nucleic Acids Res., 7, 1513 (1979)]. This plasmid was designated aspAGEL106, and its structure was confirmed by restriction enzymedigestion.

(2) Construction of pASLB3-3-1 (see FIG. 2)

According to the method described below, expression plasmid pASLB3-3-1of human granulocyte colony stimulating factor (hG-CSF) having promoterin which SV40 early gene promoter and a part of R region and U5 regionof long terminal repeat (LTR) of HTLV-1 are fused was constructed.

0.5 μg of pAGEL106 obtained in Sec. 1(1) of this Example was dissolvedin 30 μl of a buffer (abbreviated as K-20 buffer hereinafter) containing10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride, 20 mM potassiumchloride and 6 mM 2-mercaptoethanol, and digested with 10 units of SmaIat 37° C. for two hours. After precipitation with ethanol, theprecipitate was dissolved in 30 μl of T4 ligase buffer, and the digestedDNA and 0.01 μl of SalI linker (5'pGGTCGACC3': manufactured byTakarashuzo) were ligated together with 175 units of T4 DNA ligase at12° C. for 16 hours. After precipitation with ethanol, the precipitatewas dissolved in 30 μl of a buffer (abbreviated as Y-175 bufferhereinafter) containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride, 175 mM sodium chloride and 6 mM 2-mercaptoethanol, anddigested with 10 units of SalI and 10 units of MluI at 37° C. for twohours. The reaction solution was subjected to agarose gelelectrophoresis to give about 1.7 kb of DNA fragment.

On the ther hand, 1 μg of pAS3-3 obtained by the method described inEP-A 0370205 was dissolved in 30 μl of Y-175 buffer, and digested with10 units of SalI and 10 units of MluI at 37° C. for 2 hours. Thereaction solution was subjected to agarose gel electrophoresis to giveabout 6.7 kb of DNA fragment.

DNA fragments thus obtained, i.e., 0.1 μg of MluI-SalI fragment (1.7 kb)derived from pAGEL106 and 0.2 μg of MluI-SalI fragment (6.7 kb) derivedfrom pAS3-3 were dissolved in 30 μl of T4 ligase buffer and ligatedtogether with 175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the above reactionsolution according to the method by Cohen et al. to obtain a kanamycinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated aspASLB3-3-1, and its structure was confirmed by restriction enzymedigestion.

(3) Construction of pASLB3-3 (see FIG. 3)

According to the method described below, in order to construct plasmidpASLB3-3 wherein ampicillin resistance gene is introduced in pASLB3-3-1,DNA fragment [XhoI-MluI fragment (2.58 kb)] containing ampicillinresistance gene of pAS3-3 was introduced between XhoI site and MluI siteof pASLB3-3-1.

1 μg of pASLB3-3-1 obtained in Sec. 1(2) of this Example was dissolvedin 30 μl of a buffer (abbreviated as Y-150 buffer) containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride, 150 mM sodium chloride and 6mM 2-mercaptoethanol and digested with 10 units of XhoI and 10 units ofMluI at 37° C. for two hours. The reaction solution was subjected toagarose gel electrophoresis to give about 7.26 kb of DNA fragment.

Separately, 1 μg of pAS3-3 was dissolved in Y-150 buffer (30 μl) anddigested with 10 units of XhoI and 10 units of MluI at 37° C. for twohours. The reaction solution was subjected to agarose gelelectrophoresis to give about 2.58 kb of DNA fragment.

DNA fragments thus obtained, i.e., 0.2 μg of XhoI-MluI fragment (7.26kb) derived from pASLB3-3-1 and 0.1 μg of XhoI-MluI fragment (2.58 kb)derived from pAS3-3 were dissolved in T4 ligase buffer (30 μl) andligated together with 175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the above reactionmixture according to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pASLB3-3,and its structure was confirmed by restriction enzyme digestion.

(4) Construction of pASLBE3-3 (see FIG. 4)

According to the method described below, dihydrofolate reductase (dhfr)expression unit was removed from pASLB3-3 to obtain plasmid pASLBE3-3wherein replication origin (oriP) of Epstein-Barr virus and EBNA-1 geneare introduced. EBNA-1 gene encodes a factor which causes replication bytrans-acting to oriP. For that use, oriP and EBNA-1 gene were isolatedfrom plasmid p220.2 wherein SmaI-HaeIII fragment containing multicloningsites derived from pUC12 [Messing et al.: Methods in Enzymology, 101, 20(1983)] is incorporated at NarI site of p201 [Bill Sugden et al.,Nature, 313, 812 (1985)]

1 μg of Plasmid p220.2 was dissolved in Y-100 buffer (30 μl) anddigested with 20 units of EcoRI at 37° C. for two hours. Afterprecipitation with ethanol, the precipitate was dissolved in 30 μl ofDNA polymerase I buffer containing 50 mM Tris-HCl (pH 7.5), 10 mMmagnesium chloride, 0.1 mM dATP (deoxyadenosine triphosphate), 0.1 mMdCTP (deoxycytidine triphosphate), 0.1 mM dGTP (deoxyguanosinetriphosphate), and 0.1 mM dTTP (deoxythymidine triphosphate), and 6units of Escherichia coli DNA polymerase I Klenow fragment was added toreact at 37° C. for 60 minutes, which resulted in conversion of 5'protruding cohesive end produced by EcoRI digestion into blunt end. Thereaction was stopped by extraction with phenol, extracted withchloroform, and precipitated with ethanol. The precipitate was dissolvedin 20 μl of T4 ligase buffer, and XhoI linker (5'pCCTCGAGG3',manufactured by Takarashuzo) (0.05 μg) and 175 units of T4 DNA ligasewere added to react at 12° C. for 16 hours. After precipitation withethanol, the precipitate was dissolved in 30 μl of Y-100 buffer, anddigested with 10 units of BamHI at 37° C. for two hours. Afterprecipitation with ethanol, the precipitate was dissolved in 30 μl ofDNA polymerase I buffer, and 6 units of Escherichia coli DNA polymeraseI Klenow fragment was added to react at 37° C. for 60 minutes, whichresulted in conversion of 5' protruding cohensive end into blunt end.The reaction was stopped by extraction with phenol, extracted withchloroform, and precipitated with ethanol. The precipitate was dissolvedin 30 μl of-Y-100 buffer, and digested with 10 units of XhoI at 37° C.for two hours. The reaction solution was subjected to agarose gelelectrophoresis to give about 4.9 kb of DNA fragment.

Separately, 1 μg of pASLB3-3 was dissolved in 30 μl of Y-100 buffer, and20 units of XhoI was added to perform digestion reaction at 37° C. fortow hours. After precipitation with ethanol, the precipitate wasdissolved in 30 μl of DNA polymerase I buffer, and 6 units ofEscherichia coli DNA polymerase I Klenow fragment was added to react 37°C. for 60 minutes, which resulted in conversion of 5' protrudingcohesive end produced by XhoI digestion into blunt end. The reaction wasstopped by extraction with phenol, extracted with chloroform,precipitated with ethanol, the precipitate was dissolved in a buffer(abbreviated as Y-0 buffer hereinafter) containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 6 mM 2-mercaptoethanol, and 20 unitsof KpnI was added to perform digestion reaction at 37° C. for two hours.The reaction solution was subjected to agarose gel electrophoresis togive about 1.3 kb of DNA fragment.

Separately, 1 μl of pAGE107 [JP-A 3-22979, Miyaji et al.:Cytotechnology, 3, 133 (1990)] was dissolved in 30 μl of Y-0 buffer, and20 units of KpnI was added to perform digestion reaction at 37° C. fortwo hours. Thereafter, sodium chloride was added to give NaClconcentration of 100 mM, and 20 units of XhoI was added to performdigestion reaction at 37° C. for two hours. The reaction solution wassubjected to agarose gel electrophoresis to give about 6.0 kb of DNAfragment.

The DNA fragments thus obtained, i.e., 0.2 μg of XhoI-BamHI (blunt end)fragment (4.9 kb) derived from p220.2, 0.1 μg of XhoI (blunt end)-KpnIfragment (1.3 kb) derived from pASLB3-3 and 0.2 μg of KpnI-XhoI fragment(6.0 kb) derived from pAGE107 were dissolved in 30 μl of T4 ligasebuffer and ligated with 175 units of T4 DNA ligase at 12° C. for 16hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This-plasmid-was designated as pASLBE3-3,and its structure was confirmed by restriction enzyme digestion.

(5) Construction of pASLBC (see FIG. 5)

According to the method described below, plasmid pASLBC was constructedwherein hG-CSF cDNA was removed from pASLB3-3 and multicloning siteswere introduced. Multicloning sites were produced using synthetic DNA.

1 μg of pASLB3-3 obtained in Sec. 1(4) of this Example was dissolved in30 μl of Y-175 buffer and digested with 20 units of SalI and 20 units ofMluI at 37° C. for two hours. The reaction solution was subjected toagarose gel electrophoresis to give about 3.1 kb of DNA fragment.

Separately, 1 μg of pASLB3-3 was dissolved in 30 μl of Y-0 buffer anddigested with 20 units of KpnI at 37° C. for two hours. Then, sodiumchloride was added to this reaction mixture to give NaCl concentrationof 150 mM, and this plasmid was digested with 20 units of MluI at 37° C.for another two hours. The reaction mixture was subjected to agarose gelelectrophoresis to give about 6.0 kb of DNA fragment.

Separately, as a liker for linking SalI cleavage site and KpnI cleavagesite, the following DNA linker was synthesized. Restriction enzymecleavage sites HindIII, EcoRV, SfiI, StuI and NotI are incorporated inthe linker. ##STR1##

The 52 mer and 44 mer single-stranded DNAs of the DNA linker weresynthesized using a DNA synthesizer model 380A (Applied Biosystems).Each 0.2 μg of the synthesized DNAs was dissolved in 20 μl of T4 kinasebuffer and phosphorylated with 30 units of T4 polynucleotide kinase at37° C. for two hours.

The DNA fragments thus obtained, i.e., 0.1 μg of SalI-MluI fragment (3.1kb) and 0.2 μg of KpnI-MluI fragment (6.0 kb) derived from pASLB3-3 weredissolved in T4 ligase buffer (30 μl). 0.01 μg of the above DNA linkerand both DNA fragments were ligated with 175 units of T4 DNA ligase at12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pASLBC,and its structure was confirmed by restriction enzyme digestion.

(6) Construction of pASLBEC (see FIG. 6)

According to the method described below, plasmid pASLBEC was constructedby removing dihydrofolate reductase (dhfr) expression unit from pASLBCand introducing oriP and EBNA-1 genes therein.

1 μg of pASLBE3-3 obtained in Sec. 1(4) of this Example was dissolved in30 μl of Y-150 buffer and digested with 20 units of MluI and 20 units ofXhoI at 37° C. for two hours. The reaction solution was subjected toagarose gel electrophoresis to give about 1.3 kb of DNA fragment.

Separately, 1 μg of pASLBE3-3 was dissolved in 30 μl of Y-0 buffer anddigested with 20 units of KpnI at 37° C. for two hours. Thereafter,sodium chloride was added to this reaction mixture to give NaClconcentration of 150 mM and this plasmid was partially digested with 5units of MluI at 37° C. for 20 minutes. The reaction solution wassubjected to agarose gel electrophoresis to give about 9.6 kb of DNAfragment.

Then, 1 μg of pASLBC obtained in Sec. 1(5) of this Example was dissolvedin 30 μl of Y-0 buffer and digested with 20 units of KpnI at 37° C. fortwo hours. Thereafter, sodium chloride was added to this reactionmixture to give NaCl concentration of 100 mM and digested with 20 unitsof XhoI at 37° C. for another two hours. The reaction solution wassubjected to agarose gel electrophoresis to give about 0.6 kb of DNAfragment.

The DNA fragments thus obtained, i.e., 0.2 μg of MluI-XhoI fragment (1.3kb) and 0.2 μg of KpnI-MluI fragment (9.6 kb) derived from pASLBE3-3 and0.05 μg of KpnI-XhoI fragment (0.6 kb) derived from pASLBC weredissolved in 30 μl of T4 ligase buffer and ligated together with 175units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pASLBEC,and its structure was confirmed by restriction enzyme digestion.

(7) Construction of pASLBEC2 (see FIG. 7)

According to the method described below, BamHI linker was introduced inStuI site in multicloning site of pASLBEC to obtain plasmid pASLBEC2. InpASLBEC2, StuI site in multicloning sites is lost.

1 μg of pASLBEC obtained in Sec. 1(6) of this Example was dissolved in30 μl of Y-100 buffer and partially digested with 5 units of StuI at 37°C. for 20 minutes. The reaction solution was subjected to agarose gelelectrophoresis to give about 11.5 kb of DNA fragment. The resulting DNAfragment was dissolved in 30 μl of T4 ligase buffer. The DNA fragmentand 0.01 μg of BamHI linker (5'pCCGGATCCGG3': manufacture byTakarashuzo) were ligated together with 175 units of T4 DNA ligase at12° C. for 16 hours. After precipitation with ethanol, the precipitatewas dissolved in 30 μl of Y-100 buffer and digested with 20 units ofBamHI at 37° C. for two hours. The reaction solution was subjected toagarose gel electrophoresis to give about 11.5 kb of DNA fragment. Theresulting DNA fragment was dissolved in T4 ligase buffer (20 μl) andligated together with 175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmnid was designated as pASLBEC2,and its structure was confirmed by restriction enzyme digestion.

(8) Construction of pAMoEC2 (see FIG. 8)

According to the method described below, plasmid pAMoEC2 was constructedwherein promoter in pASLBEC2 [promoter in which SV40 early gene promoterand a part of R region and U5 region of long terminal repeat (LTR) ofHTLV-1 are fused] was replaced by promoter of long terminal repeat (LTR)of Moloney murine leukemia virus. For that use, Moloney murine leukemiavirus LTR promoter was isolated from plasmid Molp-1 [Akinori Ishimoto etal.: Virology, 141, 30 (1985)].

1 μg of pASLBEC2 obtained in Sec. 1(7) of this Example was dissolved ina buffer (abbreviated as K-50 buffer hereinafter) containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride, 50 mM potassium chloride and6 mM 2-mercaptoethanol and digested with 20 units of HindIII and 20units of AatII (manufactured by Toyoboseki) at 37° C. for two hours. Thereaction solution was subjected to agarose gel electrophoresis to giveabout 4.8 kb of DNA fragment.

Separately, 1 μg of pASLBEC2 was dissolved in 30 μl of K-50 buffer anddigested with 20 units of AatII at 37° C. for two hours. Thereafter,this plasmid was partially digested with 5 units of XhoI at 37° C. for20 minutes. The reaction solution was subjected to agarose gelelectrophoresis to give about 6.1 kb of DNA fragment.

Then, as a linker for linking XhoI cleavage site and ClaI cleavage site,the following linker was synthesized.

    5'TCGAGGACC3' (9 mer)

    3'CTGGGC5' (7 mer)

The 9 mer and 7 mer single-stranded DNAs of the above DNA liker weresynthesized using a DNA synthesizer model 380A (Applied Biosystems).Each 0.2 μg of the synthesized DNAs was dissolved in 40 μl of T4 kinasebuffer and phosphorylated with 30 units of T4 polynucleotide kinase at37° C. for two hours.

Separately, 1 μl of Molp-1 [Akinori Ishimoto et al.: Virology, 141, 30(1985)] was dissolved in 30 μl of K-20 buffer and digested with 20 unitsof SmaI at 37° C. for two hours. Thereafter, sodium chloride was addedto this reaction mixture to give NaCl concentration of 50 mM anddigested with 20 units of ClaI at 37° C. for two hours. The reactionsolution was subjected to agarose gel electrophoresis to give about 0.6kb of DNA fragment. The resulting DNA fragment was dissolved in 30 μl ofT4 ligase buffer. The DNA fragment, 0.01 μg of the above DNA linker and0.03 μg of HindIII linker (5'pCAAGCTTG3': manufactured by Takarashuzo)were ligated together with 175 units of T4 DNA ligase at 12° C. for 16hours. After precipitation with ethanol, the precipitate was dissolvedin 30 μl of a buffer (abbreviated as Y-50 buffer hereinafter) containing10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride, 50 mM sodium chlorideand 6 mM 2-mercaptoethanol and digested with 10 units of HindIII at 37°C. for two hours. Thereafter, sodium chloride was added to this reactionmixture to give NaCl concentration of 100 mM and digested with 10 unitsof XhoI at 37° C. for another two hours. The reaction solution wassubjected to agarose gel electrophoresis to give about 0.6 kb of DNAfragment.

The DNA fragments thus obtained, i.e., 0.2 μg of HindIII-AtaII fragment(4.8 kb) and 0.2 μg of AatII-XhoI fragment (6.1 kb) derived frompASLBEC2 and 0.05 μg of HindIII-XhoI fragment (0.6 kb) derived fromMolp-1 were dissolved in 30 μl of T4 ligase buffer and ligated togetherwith 175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmidwas-isolated-according to the known method. This plasmid was designatedas pAMoEC2, and its structure was confirmed by restriction enzymedigestion.

(9) Construction of pAMoEC3 (see FIG. 9)

According to the method described below, plasmid pAMoEC3 was constructedby inserting, as stuffer DNA, DNA fragment [DraI-PvuII fragment (2.5kb)] containing tetracycline resistant gene of pBR322 into BamHI site inmulticloning sites of pAMoEC2.

1 μg of pAMoEC2 obtained in Sec. 1(8) of this Example was dissolved in30 μl of Y-100 buffer and digested with 20 units of BamHI at 37° C. fortwo hours. After precipitation with ethanol, the precipitate wasdissolved in 30 μl of DNA polymerase I buffer, and 6 units ofEscherichia coli DNA polymerase I Klenow fragment was added to react at37° C. for 60 minutes, which resulted in conversion of 5' protrudingcohensive end produced by BamHI into blunt end. The reaction solutionwas subjected to agarose gel electrophoresis to give about 11.5 kb ofDNA fragment.

Separately, 1 μg of pBR322 [Bolivar et al.: Gene, 2, 95 (1977)] wasdissolved in 30 μl of Y-50 buffer and digested with 20 units of DraI and20 units of PvuII at 37° C. for two hours. The reaction solution wassubjected to agarose gel electrophoresis to give about 2.5 kb of DNAfragment.

The DNA fragments thus obtained, i.e., 0.1 μg of BamHI (blunt end)fragment (11.5 kb) derived from pAMoEC2 and 0.2 μg of DraI-PvuIIfragment (2.5 kb) derived from pBR322 were dissolved in 30 μl of T4ligase buffer and ligated together with 175 units of T4 DNA ligase at12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillin andtetracycline resistant strain. From this transformant, a plasmid wasisolated according to the known method. This plasmid was designated aspAMoEC3, and its structure was confirmed by restriction enzymedigestion.

(10) Construction of pAMoERC3 (see FIG. 10)

According to the method described below, plasmid pAMoERC3 wasconstructed by inverting the orientation of oriP and EBNA-1 gene inplasmid pAMoEC3.

1 μg of pAMoEC3 obtained in Sec. 1(9) of this Example was dissolved in30 μl of Y-100 buffer and digested with 20 units of XhoI at 37° C. fortwo hours. Thereafter, this plasmid was added to 30 μl of 1M Tris-HCl(pH 8.0) and dephosphorylated with one unit of Escherichia coli alkalinephosphatase (manufactured by Takarashuzo) at 37° C. for two hours. Afterprecipitation with ethanol, the precipitate was dissolved in 30 μl of abuffer (abbreviated as TE buffer hereinafter) containing 10 mM Tris-HCl(pH 8.0) and 1 mM EDTA, and the mixture was subjected to agarose gelelectrophoresis to give about 9.1 kb of DNA-fragment.

Separately, 1 μg of pAMoEC3 was dissolved in Y-100 buffer (30 μl) anddigested with 20 units of XhoI at 37° C. for two hours. The reactionsolution was subjected to agarose gel electrophoresis to give about 4.9kb of DNA fragment.

The DNA fragments thus obtained, i.e., 0.1 μg of XhoI fragment (9.1 kb)and 0.2 μg of XhoI fragment (4.9 kb) derived from pAMoEC3 were dissolvedin 30 μl of T4 ligase buffer and ligated together with 175 units of T4DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAMoERC3,and its structure was confirmed by restriction enzyme digestion.

(11) Construction of pAGE207 (see FIG. 11)

According to the method described below, plasmid pAGE207 wherein G418resistance gene in pAGE107 is replaced by hygromycin (hyg) resistancegene was constructed. For that use, hyg resistance gene was isolatedfrom p201 [Bill Sugden et al., Nature, 313, 812 (1985)].

1 μg of pAGE107 obtained by the method described in JP-A 3-22979 wasdissolved in 30 μl of Y-50 buffer and digested with 20 units of claI at37° C. for two hours. Thereafter, sodium chloride was added to thisreaction mixture to give NaCl concentration of 150 mM and digested with20 units of MluI at 37° C. for another two hours. The reaction solutionwas subjected to agarose gel electrophoresis to give about 4.6 kb of DNAfragment.

Separately, 0.5 μg of p201 [Bill Sudgen et al.: Nature, 313, 812 (1985)]was dissolved in 30 μl of Y-50 buffer and digested with 20 units of NarI[manufactured by New England Biolab] at 37° C. for two hours. Afterprecipitation with ethanol, the precipitate was dissolved in DNApolymerase I buffer (30 μl), and 6 unites of Escherichia coli DNApolymerase I Klenow fragment was added to react at 37° C. for 60minutes, which resulted in conversion of 5' protruding cohensive endproduced by NarI digestion into blunt end. The reaction was stopped byextraction with phenol, extracted with chloroform, precipitated withethanol, the precipitate was dissolved in 20 μl of T4 ligase buffer. TheDNA and 0.05 μg of ClaI linker (5'pCATCGATG3': manufactured byTakarashuzo) were ligated together with 175 units of T4 DNA ligase at12° C. for 16 hours. After precipitaiton with ethanol, the precipitatewas dissolved in 30 μl of Y-50 buffer and digested with 10 units of ClaIat 37° C. for two hours. Thereafter, sodium chloride was added to thisreaction mixture to give NaCl concentration of 150 mM and digested with10 units of MluI at 37° C. for another two hours. The reaction solutionwas subjected to agarose gel electrophoresis to give about 1.6 kb of DNAfragment.

The DNA fragments thus obtained, i.e., 0.2 μg of ClaI-MluI fragment (4.6kb) derived from pAGE107 and 0.1 μg of ClaI-MluI fragment (1.6 kg)derived from p201 were dissolved in 30 μl of T4 ligase buffer andligated together with 175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAGE207,and its structure was confirmed by restriciton enzyme digestion.

(12) Construction of pAGE207ScN (see FIG. 12)

According to the method described below, in order to remove the similarsequence with SfiI site present in rabbit βglobin gene, plasmidpAGE207ScN in which ScaI linker is inserted at Bali site of pAGE207 wasconstructed. In pAGE207ScN, the number of inserted ScaI linkers isindefinite.

0.5 μg of pAGE207 obtained in Sec. 1(11) of this Example was dissolvedin 30 μl of Y-0 buffer and digested with 10 units of BalI at 37° C. fortwo hours. After precipitation with ethanol, the precipitate wasdissolved in 20 μl of T4 ligase buffer. The DNA and 0.01 μg of ScaIlinker (5'pAAGTACTT3: manufactured-by Takarashuzo) were ligated togetherwith 175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method by Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated aspAGE207ScN, and its structure was confirmed by restriction enzymedigestion.

(13) Construction of Plasmid pAMoC3Sc (see FIG. 13)

According to the method as described below, plasmid pAMoERC3Sc wasconstructed, in which case for the purpose of removing a similarsequence with SfiI site of rabbit βglobin gene in the plasmid pAMoERC3,the rabbit βglobin gene in the plasmid pAMoERC3 was replaced with therabbit βglobin gene in the plasmid pAGE207ScN from which that similarsequence had already been removed. For convenience, plasmid pAMoC3Sc wasfirst constructed, and then the plasmid pAMoERC3Sc was constructed. Thenumber of ScaI linkers inserted into the plasmid pAGE207ScN to removethe similar sequence with SfiI site is unknown. In the case ofpAMoERC3Sc, however, from the fact that the pAGE207ScN was once digestedwith ScaI at the time of its construction, it is deduced that the numberof ScaI sites inserted thereinto is only one.

First, 1 μg of the pAGE207ScN obtained in Sec. 1(12) of this Example wasdissolved in 30 μl of Y-0 buffer and digested with 20 units of KpnI at37° C. for 2 hours. Then, sodium chloride was added to give an NaClconcentration of 100 mM, and this plasmid was further digested with 20units of ScaI at 37° C. for 2 hours. The reaction mixture was subjectedto agarose gel electrophoresis to give an about 0.7 kb DNA fragment.

Also, 1 μg of the pAGE207ScN was dissolved in 30 μl of Y-100 buffer anddigested with 20 units of ScaI and 20 units of ClaI at 37° C. for 2hours. The reaction mixture was subjected to agarose gel electrophoresisto give an about 0.9 kb DNA fragment.

Separately, 1 μg of the pAMoERC3 obtained in Sec. 1(10) of this Examplewas dissolved in 30 μl of Y-0 buffer and digested with 20 units of KpnIat 37° C. for 2 hours. Then, sodium chloride was added to give an NaClconcentration of 100 mM, and this plasmid was further digested with 20units of XhoI at 37° C. for 2 hours. The reaction mixture was subjectedto agarose gel electrophoresis to give an about 3.2 kb DNA fragment.

Next, 1 μg of the pAGE107 obtained by the method described in the EP-A0370205 was dissloved in 30 μl of Y-100 buffer and digested with 20units of XhoI and 20 units of ClaI at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis to give an about4.3 kb DNA fragment.

The DNA fragments thus obtained, i.e., 0.1 μg of the KpnI-ScaI fragment(0.7 kb) derived from the pAGE207ScN, 0.1 μg of the scaI-ClaI fragment(0.9 kb) derived from the same plasmid, 0.3 μg of the KpnI-XhoI fragment(3.2 kb) derived from the pAMoERC3, and 0.3 μg of the XhoI-ClaI fragment(4.3 kb) derived from the pAGE107 were dissolved in 30 μl of T4 ligasebuffer and ligated together with 175 units of T4 DNA ligase at 12° C.for 16 hours.

Escherichia coli HB101 strain was transformed using the reaction mixtureaccording to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAMoC3Sc,and its structure was confirmed by restriction enzyme digestion.

(14) Construction of Plasmid pAMoERC3Sc (see FIG. 14)

First, 1 μg of the pAMoERC3 obtained in Sec. 1(10) of this Example wasdissloved in 30 μl of Y-0 buffer and digested with 20 units of KpnI at37° C. for 2 hours. Then, sodium chloride was added to give an NaClconcentration of 150 mM, and this plasmid was further digested with 20units of MluI at 37° C. for 2 hours. The reaction mixture was subjectedto agarose gel electrophoresis to give an about 6.8 kb DNA fragment.

Also, 1 μg of the pAMoERC3 was dissolved in 30 μl of Y-150 buffer anddigested with 20 units of XhoI and 20 units of MluI at 37° C. for 2hours. The reaction mixture was subjected to agarose gel electrophoresisto give an about 1.3 kb of DNA fragment.

Separately, 1 μg of the pAMoC3Sc was dissloved in 30 l of Y-0 buffer anddigested with 20 units of KpnI at 37° C. for 2 hours. Then, sodiumchloride was added to this reaction mixture to give an NaClconcentration of 100 mM, and this plasmid was digested with 20 units ofXhoI at 37° C. for 2 hours. The reaction mixture was subjected toagarose gel electrophoresis to give an about 5.9 kb DNA fragment.

The DNA fragments thus obtained, i.e., 0.2 μg of the KpnI-MluI fragment(6.8 kb) derived from the pAMoERC3, 0.05 μg of the XhoI-MluI fragment(1.3 kb) derived from the same plasmid, and 0.2 μg of the KpnI-XhoIfragment (5.9 kb) derived from the pAMoC3Sc Were dissloved in 30 μl ofT4 ligase buffer and ligated together with 175 units of T4 DNA ligase at12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated aspAMoERC3Sc, and its structure was confirmed by restriction enzymedigestion.

The plasmid pAMoERC3Sc has a long terminal repeat of Moloney murineleukemia virus as a promoter for expression of heterogeneous genes. Forthe purpose of attaining high efficiency in the expression ofheterogeneous genes, this plasmid is desighed to have a rabbit βglobingene splicing signal, a rabbit βglobin gene poly A addition signal, andan SV40 early gene poly A addition signal after the position of aheterogeneous genes to be inserted. Moreover, this plasmid has a G418resistance gene as a drug resistance marker for animal cells and akanamycin resistance gene (the same as the G418 resistance gene) and anampicillin resistance gene as drug resistant markers for Escherichiacoli cells, respectively. Further, this plasmid has a replication origin(oriP) of Epstein-Barr viruses and an EBNA-1 gene which is atrans-acting factor on the oriP to cause replication, so that it can bepresent in many kinds of cells, including Namalwa cells other thanrodent cells, in the form of a plasmid without being incorporated intotheir chromosomes.

2. Resistance of Namalwa cells to Ricinus communis 120 lectin

Namalwa cells conditioned for serum-free media (KJM-1 strain) (Hosoi etal., Cytotechnology, 1, 151 (1988)) were cultured in the presence ofRicinus communis 120 lectin at various concentrations, and theresistance of the KJM-1 strain to Ricinus communis 120 lectin wasexamined. The KJM-1 strain was suspended in RPMI1640.ITPSGF medium(RPMI1640 medium (Nissui Seiyaku) containing a 1/40 volume of 7.5%NaHCO₃, 200 mM L-glutamine solution (GIBCO) of 3% in volume,penicillin-streptomycin solution (GIBCO; 5000 units/ml penicillin and5000 μg/ml streptomycin) of 0.5% in volume, 10 mM HEPES, 3 μg/mlinsulin, 5 μg/ml transferrin, 5 mM sodium pyruvate, 125 nM sodiumselenate, 1 mg/ml galactose, and 0.1% (w/v) prulonic F68) to give aconcentration of 5×10⁴ cells/ml, and the suspension was distributed in200-μl portions into wells of a 96-well microtiter plate. Variousconcentrations of Ricinus communis 120 lectin (Seikagaku Kogyo) wereadded thereto in 1/100 volumes, and the plate was incubated in a CO₂incubator at 37° C. for 1 to 2 weeks. As the result, it was found thatthe minimum concentration of Ricinus communis 120 lectin to causecomplete inhibition of the KJM-1 strain growth was 50 ng/ml. Fourmillion cells of the KJM-1 strain were examined, and the naturaloccurrence of Ricinus communis 120 lectin resistant strain was notobserved.

3. Cloning of α2→3 sialyltransferase cDNA (LEC) from human histiocyticleukemia cell line TYH

(1) Isolation of mRNA from TYH cells

From 1×10⁸ TYH cells (Haranaka et al., Int. J. Cancer, 36, 313 (1985)),about 40 μg of mRNA was isolated using the mRNA extraction kit "FastTrack" (Invitrogen; trade No. K1593-02).

(2) Preparation of cDNA library (see FIG. 15)

From 8 μg of mRNA obtained above, double-stranded cDNA was preparedusing the cDNA synthesis kit "The Librarian I" (Invitrogen) with arandom primer as a primer. Then, each of the SfiI linkers (Seq. ID: 5)as shown below was provided, instead of BstXI linkers, at eitherterminus of the cDNA. The cDNA-was fractioned in size by agarose gelelectrophoresis and cDNA fragments larger than about 1.2 kb wereisolated.

SfiI Linkers:

    5'-CTTTAGAGCAC-3' (11 mer)

    3'-GAAATCTC-5' (8 mer)

The 11 mer and 8 mer single-stranded DNAs of SfiI linkers weresynthesized using a DNA synthesizer model 380A (Applied Biosystems).Then, 50 μg of each of the synthesized DNAs was dissloved in 50 μl of T4kinase buffer and phosphorylated with 30 units of T4 polynucleotidekinase (Takara Shuzo) at 37° C. for 16 hours. The specific reagents andprocedures were as described in the manufacturer's instructionsaccompanying the kit used, except that the above SfiI linkers were usedin place of BstXI linkers.

Also, 24 μg of pAMoERC3Sc, which is a direct expression cloning vector,was dissloved in 590 μl of Y-50 buffer and digested with 80 units ofSfiI at 37° C. for 16 hours. Then, 5 μl of this solution was subjectedto agarose gel electrophoresis, and after the completion of digestionwas confirmed, for the purpose of decreasing the amount of clones havingno cDNA insert-incorporated at the time of cDNA library construction,this plasmid was further digested with 40 units of BamHI at 37° C. for 2hours. The reaction mixture was subjected to electrophoresis to giveabout 11.5 kb DNA fragment.

Next, 2 μg of the SfiI fragment (11.5 kb) derived from the pAMoERC3Sc,together with the cDNA obtained above, was dissolved in 250 μl of T4ligase buffer and ligated together with 2000 units of T4 DNA ligase at12° C. for 16 hours. Then, 5 μg of transfer RNA (tRNA) was addedthereto, followed by ethanol precipitation, and the resultingprecipitate was dissolved in 20 μl of TE buffer. With this reactionmixture, Escherichia coli strain LE392 (Maniatis et al., ed., MolecularCloning, 2.58, Cold Spring Harbor, 1989) was tranformed byelectroporation (William J. Dower et al., Nucleic Acids Res., 16, 6127(1988)) to give about 200,000 ampicillin resistant transformants.

(3) Cloning of α2→3 sialyltransferase cDNA (LEC)

About 200,000 ampicillin-resistant transformants (cDNA library) obtainedin Sec. 3(2) of this Example were mixed, after which a plasmid wasprepared using >plasmid<maxi kit (trade No. 41031; Qiagen) which is aplasmid preparation kit. The obtained plasmid was ethanol precipitated,and the resulting precipitate was dissolved in TE buffer to give aconcentration of 1 μg/μl.

The above plasmid was introduced into the KJM-1 strain byelectroporation (Miyaji et al., Cytotechnology, 3, 133 (1990)) at aproportion of 4 μg per 1.6×10⁶ cells. After the introduction of plasmid,these cells were suspended in 8 ml of RPMI1640.ITPSGF medium, and thecells were incubated in a CO₂ incubator at 37° C. for 24 hours. Then,the cells were supplemented with G418 (GIBCO) to give a concentration of0.5 mg/ml and further cultured for 5 to 7 days to give transformants.The obtained transformants were suspended in RPMI1640.ITPSGF mediumcontaining Ricinus communis 120 lectin (50 ng/ml) to give aconcentration of 5×10⁴ cells/ml, and the cells were distributed in200-μl portions into wells of a 96-well microtiter plate. The cells werecultured in a CO₂ incubator at 37° C. for 2 to 3 weeks, and 7 strainswere obtained which had become resistant to a Ricinus communis 120lectin. From this resistant strain, a plasmid was isolated according tothe Hirt method (Robert F. Margolskee et al., Mol. Cell. Biol., 8, 2837(1988)), and Escherichia coli strain LE392 was transformed with thisplasmid by electroporation (Willium J. Dower et al., Nucleic Acids Res.,16, 6127 (1988)). From this transformant, a plasmid was preparedusing >plasmid<maxi kit (trade No. 41031; Qiagen), and its structure wasexamined by restriction enzyme digestion to find that it contained about1.9 kb cDNA. The cDNA containing plasmid was designated as pAMoERL. Whenthis plamid was also introduced into the KJM-1 strain by the abovemethod, this strain became resistant to Ricinus communis 120 lectin; itwas therefore found that this cDNA is a gene responsible for lectinresistance. The KJM-1 strain containing the plasmid pAMoERL was able togrow even in the presence of 200 ng/ml of Ricinus communis 120 lectin.

4. Sequensing of α2→3 sialyltransferase cDNA (LEC)

(1) Introduction of α2→3 sialyltransferase cDNA (LEC) into pUCl19 (seeFIG. 16)

First, 1 μg of pAMoERL obtained in Sec. 3(3) of this Example wasdissloved in 30 μl of Y-100 buffer and digested with 20 units of ECoRVand 20 units of Asp718 (Boehringer Mannheim) at 37° C. for 2 hours. Thereaction mixture was subjected to electrophoresis to give an about 1.97kb DNA fragment.

Separately, 1 μg of pUCl19 (Messing et al., Methods in Enzymology, 153,3 (1987)) was dissloved in 30 μl of K-20 buffer and digested with 20units of SmaI at 37° C. for 2 hours. Then, sodium chloride was added togive an NaCl concentration of 100 mM, and this plasmid was furtherdigested with 20 units of Asp718 at 37° C. for 2 hours. The reactionmixture was subjected to electrophoresis to give an about 3.16 kb DNAfragment.

The DNA fragments thus obtained, i.e., 0.2 μg of the ECoRV-Asp718fragment (1.97 kb) derived from the pAMoERL and 0.1 μg of theSmaI-Asp718 fragment (3.16 kb) derived from the pUCl19 were dissloved in30 μl of T4 ligase buffer and ligated together with 175 units of T4 DNAligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated aspUCl19-LEC, and its structure was confirmed by restriction enzymedigestion.

(2) Construction of deletion plasmids for sequencing

First, 2 μg of pUCl19-LEC obtained in Sec. 4(1) of this Example wasdissloved in 30 μl of Y-150 buffer and digested with 20 units of BamHIand 20 units of SphI at 37° C. for 2 hours. After ethanol precipitation,the resulting precipitate was dissolved in 100 μl of ExoIII buffer(deletion kit for kilo-sequence; Takara Shuzo). Also, 2 μg of pUCl19-LECwas dissloved in 30 μl of Y-0 buffer and digested with 20 units of SacIat 37° C. for 2 hours. Then, sodium chloride was added to give an NaClconcentration of 150 mM, and this plasmid was further digested with 20units of NotI at 37° C. for 2 hours. After ethanol precipitation, theresulting precipitate was dissloved in 100 μl of ExoIII buffer.

From the BamHI-SphI fragment and the SacI-NotI fragment thus obtainedfrom the pUCl19-LEC, several tens of deletion plasmids were prepared,respectively, using the deletion kit for kilo-sequence (Takara Shuzo).

The nucleotide sequence of the deletion plasmid obtained above wasdetermined using the Taq DyeDeoxy terminator cycle sequencing kit (tradeNo. 401113; Applied Biosystems). The determined nucleotide sequence isshown in the Sequence Listing (Seq. ID: 1). Also, it was found from thecorresponding-amino acid sequence that this protein has a commonstructure to glycosyltransferase (hereinafter abbreviated to GT). Thatis, this protein seems to have a structure where 8 amino acids in theN-terminal portion are put out at the cytoplasm side, a highlyhydrophobic region consisting of the subsequent 18 amino acids is usedfor binding to the membrane, and most of the remaining C-terminalportion including -the catalytic site is exposed to the internal cavityof the Golgi's apparatus. The comparison of amino acid sequence betweenthis protein and other GTs made clear that this protein has a certainhomology with rat α2→6 sialyltransferase. For these reasons, it isconsidered that Ricinus communis 120 lectin resistance gene encodes GT.

5. Measurement of α2→3 sialyltrasferase activity of the KJM-1 strainhaving expression plasmid for Ricinus communis 120 lectin resistancegene

The KJM-1 strain having the plasmid pAMoERL obtained in Sec. 3 of thisExample was suspended in 30 ml of RPMI1640.ITPSGF medium containing 0.5mg/ml of G418 to give a concentration of 5×10⁵ cells/ml, and the cellswere cultured in a CO₂ incubator at 37° C. for 3 days. After theculturing, the cells were collected by centrifugation at 160×g for 10minutes and washed with 10 ml of PBS (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/lanhydrous sodium monohydrogenphosphate, 0.2 g/l potassiumdihydrgenphosphate), followed by further centrifugation to collect thecells.

About 1.4×10⁷ cells obtained above were suspended in 100 μl ofhomogenization buffer (250 mM saccarose, 10 mM Tris-HCl (pH 7.4)), andlysed by sonication. The lysate was cetrifuged at 550×g for 10 minutesto obtain a supernatant.

Also, as a control, the KJM-1 strain having the vector plasmidpAMoERC3Sc was prepared, and the above procedures were followed toobtain a supernatant.

Then, 20 μl of each of the two supernatants obtained above was allowedto react in an assay solution (0.1M cacodylic acid-HCl (pH 6.5), 0.01MMnCl₂, 0.45% Triton X-100, 0.08 mM substrate, 5 mM CMP-sialic acid(added or not added)) having the final volume of 50 μl at 37° C. for 2hours, and the products were identifed by high performance liquidchromatography (HPLC) to determine the α2→3 sialyltransferase activityin the respective supernatants. The activity determinations were carriedout using 200 μg of proteins in the supernatant, and proteinquantitation was achieved using BCA protein assay reagent (PIERCE). Asthe substrate, a sugar chain fluorescence-labeled with aminopyridine(Galβ1→4GlcNAcβ1→3Galβ1→4Glc-aminopyridine) was used, The fluorescencelabeling of the substrate was carried out using lacto-N-neotetraose(BioCarb Chemicals) by the conventional method (Akimoto Kondo, et al.,Agric. Biol. Chem., 54, 2169 (1990)). Each of the supernatants wasallowed to react with an assay solution containing or not containingCMP-sialic acid as a sugar donor. The reaction mixture was separated byHPLC, and the peaks appearing only with the assay solution containingCMP-sialic acid were considered as the products. After completion of thereaction, the assay solution was treated at 100° C. for 5 minutes, andcentrifuged at 10,000×g for 10 minutes. The resulting supernatant wasSubjected to HPLC which was carried out on the TSKgel ODS-80T_(M) column(4.6 mm×30 cm; Tosoh) eluting with 0.02M ammonium acetate buffer (pH4.0) at a temperature of 50° C. at a rate of 1 ml/min. The products weredetected using the fluorescence HPLC monitor model RF-535T (ShimazuSeisakusho) with an excitation wavelength of 320 nm and an emissionwavelength of 400 nm. As the result, peaks 1 and 2 were detected as theproducts, as shown in FIG. 17. From the facts that the elution time wasidentical with that of the standard and that the substrate wasregenerated by sialidase treatment of the products, it was found thatthe peak 1 corresponds toNeuAcα2→6Galβ1→4GlcNAcβ1→3Galβ1.fwdarw.4Glc-aminopyridine and the peak 2corresponds toNeuAcα2→3Galβ1→4GlcNAcβ1→3Galβ1.fwdarw.4Glc-aminopyridine.

The sialidase treatment of the products was carried out as follows.First, 10 μl of the supernatant was subjected to HPLC, and the peaks 1and 2 were fractioned and freeze-dried, independently, followed bydissolution in 50 μl of buffer containing 20 mM Tris-maleic acid (pH6.0) and 1 mM calcium citrate. Then, 20 μl of the solution was treatedwith 2 μl of 400 mU/ml sialidase (neuraminidase; Sigma, N-2133) at 37°C. for 16 hours. Also, as a control, the same reaction was conductedusing 2 μl of water in place of sialidase. After completion of thereaction, the solution was treated at 100° C. for 5 minutes, andcentrifuged at 10,000×g for 10 minutes. Then, 10 μl of the supernatantwas subjected to the above HPLC. The results are shown in FIG. 18. Bothfrom the peak 1 and from the peak 2, peak 3 was detected as the productby sialidase treatment. In view of its elution time, the peak 3 isconsidered to be Galβ1→4GlcNAcβ1→3Galβ1→4Glc-aminopyridine as thesubstrate.

The comparison of HPLC pattern between the KJM-1 strain having theplasmid pAMoERL and the KJM-1 strain having the plasmid pAMoERC3Sc madeclear that both the strains gave approximately the same peak 1 but thestrain having the pAMoERL strain gave a significantly higher peak 2 thanthat of the strain having the pMoERC3Sc. The ratio of peak 2 to peak 1for the KJM-1 strain having the pAMoERL was 6 to 7 times greater thanthat for the KJM-1 strain having the pAMoERC3Sc as the vector (see FIG.17). From these results, it was shown that this Ricinus communis 120lectin resistance gene is an α2→3 sialyltransferase gene and thatoligosaccharides with sialic acid added can be produced using α2→3sialyltransferase encoded in the said gene.

EXAMPLE 2

Synthesis of sialyl-Le^(x) in the strain KJM-1 having the expressionplasmid for α2→3 sialyltransferase:

The KJM-1 strain having the plasmid pAMoERL obtained in Sec. 3(3) ofExample 1 and the strain KJM-1 having the direct expression cloningvector pAMoERC3Sc obtained in Sec. 1(14) of Example 1 were independentlycultured in the RPMI1640.ITPSGF medium containing 0.5 mg/ml of G418.Then, about 1×10⁶ cells of each strain were taken in a microtube (1.5ml; Eppendorf) and centrifugated at 550×g for 7 minutes. The collectedcells were washed with 1 ml of PBS containing 0.1% sodium azide(hereinafter abbreviated as A-PBS), and the expression of sialyl-Le^(x)in the cells of each strain was examined by indirect fluorescentantibody staining with KM93 (Shitara et al., Anticancer Res., 9, 999(1989)) which is a monoclonal antibody reacting with sialyl-Le^(x), asdescribed below.

The cells of each strain were suspended in 50 μl of A-PBS solutioncontaining 10 μg/ml KM93 and allowed to react at 4° C. for 1 hour. Afterwashing three times with A-PBS, these cells were suspended in 20 μl ofA-PBS containing the anti-mouse antibodies IgG and IgM (Cappel) diluted20-fold with A-PBS which had been fluorescence-labeled with fluoresceinisothiocyanate (FITC), and allowed to react at 4° C. for 30 minutes.After washing three times with A-PBS, these cells were again suspendedin A-PBS, and analysis was carried out with the flow cell sorter FCS-1(Nihon Bunko). As a control, the same analysis was carried out using thenormal mouse serum diluted 500-fold with A-PBS in place of KM93. Theresults are shown in FIG. 19. The fluorescence intensity of the cellsstained with KM93 for the KJM-1 strain having the direct expressioncloning vector pAMoERC3Sc was stronger than that of the control (seeFIG. 19(a)). This indicates that sialyl-Le^(x) is also expressed in theoriginal KJM-1 strain. The fluorescence intensity of the cells stainedwith KM93 for the KJM-1 strain having the plasmid pAMoERL capable ofexpressing cDNA encoding α2→3 sialyltrasferase of the present inventionwas further stronger than that of the strain KJM-1 containing thepAMoERC3Sc (see FIG. 19(b)). This indicates that sialyl-Le^(x) issynthesized in cells by α2→3 sialyltransferase of the present invention.

EXAMPLE 3

Production by animal cells of α2→3 sialyltransferase derived from TYHcells:

1. Construction of plasmid pAMoPRSAL-35F for expression of cDNA encodingα2→3 sialyltransferase

(1) Construction-of pAGE147 (see FIG. 20)

According to the method as described below, the plasmid pAGE147 wasconstructed by replacing the SV40 early gene promoter of the plasmidpAGE107 with a long terminal repeat (LTR) of Moloney murine leukemiavirus as a promoter.

First, 2 μg of the plasmid pPMOL1 obtained by the method as described inJP-A 1-63394 was dissloved in 30 μl of Y-0 buffer and digested with 20units of SmaI at 30° C. for 3 hours. Then, sodium chloride was added togive an NaCl concentration of 50 mM, and this plasmid was furtherdigested with 20 units of ClaI at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis to give an about0.6 kb DNA fragment containing the LTR promoter of Moloney murineleukemia virus.

Next, 25 pmoles of each of the two synthetic DNA fragments as shownbelow, which had been synthesized in Sec. 1(8) of Example 1, weredissloved in 10 μl of T4 kinase buffer and phospholylated at their5'-termini with 5 units T4 DNA kinase at 37° C. for 30 minutes.

    5'-TCGAGGACC-3' (9 mer)

    3'-CCTGGGC-5' (7 mer)

The DNA fragments thus obtained, i.e., 0.05 μg of the ClaI-SmaI fragment(0.6 kb) derived from the pPMOL1, two 5'-phospholylated synthetic DNAfragments (each 1 pmol), and HindIII linker (5'-pCAAGCTTG-3'; TakaraShuzo) (1 pmol) were dissloved in 30 μl of T4 ligase buffer and ligatedtogether with 200 units of T4 DNA ligase at 12° C. for 16 hours. Afterrecovery by ethanol precipitation, the DNA fragments were dissloved inY-100 buffer and digested with 10 units of HindIII and 10 units of XhoIat 37° C. for 2 hours. The reaction was stopped by phenol/chloroformextraction and the DNA fragments were recovered by ethanolprecipitation.

Independently, 1 μg of pAGE107 (JP-A 3-22979; Miyaji et al.,Cytotechnology, 3, 133 (1990)) was dissloved in 30 μl of Y-100 bufferand digested with 10 units of HindIII and 10 units of XhoI at 37° C. for2 hours. The reaction mixture was subjected to agarose gelelectrophoresis to give an about 6.0 kb DNA fragment containing the G418resistance gene and ampicillin resistance gene.

The DNA fragments thus obtained, i.e., 0.3 μg of the HindIII-XhoIfragment (6.0 kb) derived from the pAGE107 and 0.01 μg of theHindIII-XhoI fragment (0.6 kb) derived from the pPMOL1 were dissloved in20 μl of T4 ligase buffer and ligated together with 200 units of T4 DNAligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was trasformed using this reaction mixtureaccording to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAGE147,and its structure was confirmed by restriction enzyme digestion.

(2) Construction of pAGE247 (see FIG. 21)

According to the method as described below, the plasmid pAGE247 wasconstructed by replacing the SV40 early gene promoter of the plasmidpAGE207 with a long terminal repeat (LTR) of Moloney murine leukemiavirus as a promoter.

First, 2 μg of the plasmid pAGE147 obtained in Sec. 1(1) of this Examplewas dissolved in 30 μl of Y-100 buffer and digested with 10 units ofHindIII and 10 units of XhoI at 37° C. for 2 hours. The reaction mixturewas subjected to agarose gel electrophoresis to give an about 0.63 kbDNA fragment containing the LTR promoter of Moloney murine leukemiavirus.

Independently, 2 μg of the plasmid pAGE207 obtained in Sec. 1(11) ofExample 1 was dissloved in 30 μl of Y-100 buffer and digested with 10units of HindIII and 10 units of XhoI at 37° C. for 2 hours. Thereaction mixture was subjected to agarose gel electrophoresis to give anabout 5.84 kb DNA fragment containing the hyg resistance gene and theampicillin resistance gene.

The DNA fragments thus obtained, i.e., 0.05 μg of the HindIII-XhoIfragment (0.63 kb) derived from the pAGE147 and 0.1 μg of theHindIII-XhoI fragment (5.84 kb) derived from the pAGE207 were disslovedin 30 μl of T4 ligase buffer and ligated together with 100 units of T4DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was trasformed using this reaction mixtureaccording to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAGE247,and its structure was confirmed by restriction enzyme digestion.

(3) Construction of pAMN6hyg (see FIG. 22)

According to the method as described below, the plasmid pAMN6hyg forexpression of human granulocyte colony stimulating factor derivative wasconstructed which contains the LTR of Moloney murine leukemia virus as apromoter and the hyg resistance gene as a marker.

First, 2 μg of the plasmid pAGE247 obtained in Sec. 1(2) of this Examplewas dissloved in 30 μl of Y-50 buffer and digested with 20 units of ClaIat 37° C. for 2 hours. Then, sodium chloride was added to give an NaClconcentration of 175 mM, and this plasmid was further digested with 20units of SalI at 37° C. for 2 hours. The reaction mixture was subjectedto agarose gel electrophoresis to give an about 4.8 kb DNA fragmentcontaining the LTR promotor of Moloney murine leukemia virus, theampicillin resistance gene and the hyg resistance gene.

Separately, 2 μg of the plasmid pASN6 obtained by the method asdescribed in EP-A 0370205 was dissolved in 30 μl of Y-50 buffer anddigested with 20 units of ClaI at 37° C. for 2 hours. Then, sodiumchloride was added to give an NaCl concentration of 175 mM, and thisplasmid was further digested with 20 units of SalI and 20 units of MluIat 37° C. for 2 hours. The reaction mixutre was subjected to agarose gelelectrophoresis to give an about 5.0 kb DNA fragment containing thehuman granulocyte colony stimulating factor derivative gene.

The DNA fragments thus obtained, i.e., 0.1 μg of the ClaI-SalI fragment(4.8 kb) derived from the pAGE247 and 0.1 μg of the CalI-SalI fragment(5.0 kb) derived from the pASN6 were dissloved in 20.7μl of T4 ligasebuffer and ligated together with 200 units of T4 DNA ligase at 12° C.for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAMN6hygand its structure was confirmed by restriction enzyme digestion.

(4) Construction of secretory expression vector pAMoERSA (see FIG. 23)

According to the methods as described below, the secretory expressionvector pAMoERSA for secretory expression of an arbitrary protein as afused protein with the binding region of Staphylococcus aureus protein Ato immunoglobulin G (IgG) was constructed,

First, 2 μg of the plasmid pAMN6hyg obtained in Sec. 1(3) of thisExample was dissloved in 30 μl of Y-50 buffer and digested with 20 unitsof SnaBI at 37° C. for 2 hours. Then, sodium chloride was added to givean NaCl concentration of 100 mM, and this plasmid was further digestedwith 20 units of XbaI at 37° C. for 2 hours. The reaction mixture wassubjected to agarose gel electrophoresis to give an about 0.33 kb DNAfragment containing the signal sequence of human granulocyte colonystimulating factor.

Also, 2 μg of the pPrAS1 (Saito et al., Protein Engineering, 2, 481(1989)) was dissloved in 30 μl of Y-50 buffer and digested with 20 unitsof ClaI at 37° C. for 2 hours. After ethanol precipitation, theprecipitate was dissloved in 30 μl of DNA polymerase I buffer, and the5'-cohesive end produced by the ClaI digestion was converted into ablunt end with 6 units of the Klenow fragment of Escherichia coli DNApolymerase I at 37° C. for 60 minutes. The reaction was stopped byphenol extraction, and after chloroform extraction and ethanolprecipitation, the resulting precipitate was dissloved in 30 μl of Y-100buffer and digested with 20 units of BamHI at 37° C. for 2 hours. Thereaction mixture was subjected to agarose gel electrophoresis to give anabout 0.21 kb DNA fragment containing the IgG-binding region ofStaphylococcus aureus protein A.

Separately, 2 μg of the plasmid pAMoERC3Sc obtained in 1(14) of Example1 was dissloved in 30 μl of Y-100 buffer and digested with 20 units ofXbaI and 20 units of BamHI at 37° C. for 2 hours. The reaction mixturewas subjetced to agarose gel electrophoresis to give an about 12.1 kbDNA fragment.

The DNA fragments thus obtained, i.e., 0.05 μg of the SnaBI-XbaIfragment (0.33 kb) derived from the pAMN6hyg, 0.05 μg of theClaI(blunt)-BamHI fragment (0.21 kb) derived from the pPrAS1, and 0.1 μgof the XbaI-BamHI fragment (12.1 kb) derived from the pAMoERC3Sc weredissloved in 30 μl of T4 ligase buffer and ligated together with 175units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the hnown method. This plasmid was designated as pAMoERSA,and its structure was confirmed by restriction enzyme digestion.

(5) Construction of pAMoPRC3Sc (see FIG. 24)

In cases where certain cells capable of expressing EBNA-1, such asNamalwa cells, are used as a host, the plasmid introduced into the hostcan be present as it is, without being integrated into the chromosome,even if it has no EBNA-1 gene in the plasmid pAMoERC3Sc. According tothe methods as described below, therefore, the plasmid pAMoPRC3Sc wasconstructed by removing the EBNA-1 gene in the plasmid pAMoERC3Sc. Theplasmid pAMoPRC3Sc can be used as a direct expression cloning vectorsimilarily to the case of the plasmid pAMoERC3Sc.

First, 2 μg of the plasmid pAMoERC3Sc obtained in Sec. 1(14) of Example1 was dissolved in 30 μl of Y-50 buffer and digested with 20 units ofNsiI (New England Biolabs) at 37° C. for 2 hours. After ethanolprecipitation, the resulting precipitate was dissolved in 30 μl of DNApolymerase I buffer, and the 3'-cohesive end produced by the NsiIdigestion was converted into a blunt end with 6 units of the Klenowfragment of Escherichia coli DNA polymerase I at 37° C. for 60 minutes.The reaction was stopped by phenol extraction, and after chloroformextraction and ethanol precipitation, the resulting precipitate wasdissloved in 30 μl of Y-100 buffer and digested with 20 units of NotI at37° C. for 2 hours. The reaction mixture was subjected to agarose gelelectrophoresis to give an about 8.1 kb DNA fragment.

Separately, 2 μg of the same plasmid as above was dissloved in 30 μl ofY-100 buffer and digested with 20 units of XhoI at 37° C. for 2 hours.After ethanol precipitation, the resulting precipitate was dissloved in30 μl of DNA polymerase I buffer, and the 5'-cohesive end produced bythe XhoI digestion was converted into a blunt end with 6 units of theKlenow fragment of Escherichia coli DNA polymerase I at 37° C. for 60minutes. The reaction was stopped by phenol extraction, and afterchloroform extraction and ethanol precipitation, the resultingprecipitate was dissloved in 30 μl of Y-100 buffer and digested with 20units of NotI at 37° C. for 2 hours. The reaction mixture was subjectedto agarose gel electrophoresis to give an about 3.2 kb DNA fragment.

The DNA fragments thus obtained, i.e., 0.1 μg of the NsiI(blunt)-NotIfragment (8.1 kb) derived from the plasmid pAMoERC3Sc and 0.1 μg of theXhoI(blunt)-NotI fragment (3.2 kb) derived from the same plasmid weredissloved in 30 μl of T4 ligase buffer and ligated together with 175units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAMoPRC3Scand its structure was confirmed by restriction enzyme digestion.

(6) Construction of pAMoPRSA (see FIG. 25)

According to the methods as described below, the plsamid pAMoPRSA wasconstructed by removing the EBNA-1 gene in the pAMoERSA. The plasmidpAMoPRSA can be used as a secretory expression vector similarily-to thecase of the plasmid pAMoERSA.

First, 2 μg of the plasmid pAMoERSA obtained in Sec. 1(4) of thisExample was dissloved in 30 μl of a buffer containing 10 mM Tris-HCl (pH7.5), 6 mM MgCl₂, 80 mM NaCl, and 6 mM 2-mercaptoethanol (hereinafterabbreviated as Y-80 buffer) and digested with 20 units of XbaI and 20units of ASp718 (Boehringer Mannheim) at 37° C. for 2 hours. Thereaction mixture was subjected to agarose gel electrophoresis to give anabout 1.3 kb DNA fragment.

Separately, 2 μg of the plasmid pAMoPRC3Sc was dissolved in 30 μl ofY-100 buffer and digested with 20 units of XbaI and 20 units;of Asp718at 37° C. for 2 hours. The reaction mixture was subjected to agarose gelelectrophoresis to give an about 8.5 kb DNA fragment.

The DNA fragments thus obtained, i.e., 0.05 μg of the XbaI-Asp718fragment (1.3 kb) derived from the pAMoERSA and 0.1 μg of theXbaI-Asp718 fragment (8.5 kb) derived from the pAMoPRC3Sc were dissolvedin 30 μl of T4 ligase buffer and ligated together with 175 units of T4DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated as pAMoPRSA,and its structure was confirmed by restriction enzyme digestion.

(7) Construction of pAMoPRSAL-35F (see FIG. 26)

The cloned α2→3 sialyltransferase seems to have a structure where 8amino acids at the N-terminal portion are put out at the cytoplasm side,the highly hydrophobic region consisting of the subsequent 18 aminoacids is used for binding to the membrane, and most of the remainingC-terminal portion including the catalytic site is exposed to theinternal cavity of Golgi's apparatus. According to the methods asdescribed below, therefore, the secretory expression of α2→3sialyltransferase was achieved by removing the membrane-binding regionfrom α2→3 sialyltransferase and adding instead the signal sequence ofgranulocyte colony stimulating factor and the IgG-binding region ofStaphylococcus aureus protein A.

The cDNA portion encoding a certain region on and after themembrane-binding region of α2→3 sialyltransferase (from 35th Phe to333rd Phe) was prepared by the polymerase chain reaction (PCR) methodand inserted into the secretory expression vector pAMoPRSA obtained inSec. 1(6) of this Example.

As a set of primers using PCR, the following two synthetic DNAfragments, i.e., L-A(35F) (44 mer) and L-3NN (36 mer), were synthesizedby DNA synthesizer model 380A (Applied Biosystems).

    L-A(35F) (44 mer): 5'-CTCTCCGATATCTGTTTTATTTTCCCATCTCAGAGAAGAAAGAG-3'

    L-3NN (36 mer): 5'-GATTAAGGTACCAAGTCAGAAGTATGTGAGGTTCTT-3'

The primers using PCR L-A(35F) and L-3NN are designed to have an EcoRVsite and an Asp718 site, respectively, so that DNA fragments amplifiedby PCR can be incorporated between the StuI site and the Asp718 site ofthe plasmid pAMoPRSA after digestion with EcoRV and Asp718. The PCR wascarried out using a GeneAmp™ DNA amplification reagent kit withAmpliTaq™ recombinant Taq DNA polymerase (Takara Shuzo). The reactionmixture was prepared accodring to the methods as described in the kit,and Perkin Elmer Cetus DNA thermal cycler (Takara Shuzo) was used forincubation. Thirty cycles of amplification were carried out according tothe following scheme: 94° C. for 1 minute, 55° C. for 1 minute and 72°C. for 3 minutes. Then, further incubation was carried out at 72° C. for7 minutes. As a template, 1 ng of the plasmid pUCl19-LEC obtained inExample 1 was used. After completion of the reaction, chloroformextraction and ethanol precipitation were successively carried out. Theresulting precipitate was then dissloved in 30 μl of Y-100 buffer anddigested with 20 units of EcoRV and 20 units of Asp718 at 37° C. for 2hours. The reaction micture was subjected to agarose gel electrophoresisto give an about 0.9 kb DNA fragment.

Separately, 2 μg of the plasmid pAMoPRSA was dissloved in 30 μl of Y-100buffer and digested with 20 units of StuI and 20 units of Asp718 at 37°C. for 2 hours. The reaction mixture was subjected to agarose gelelectrophoresis to give an about 9.06 kb DNA fragment.

The DNA fragments thus obtained. i.e., 0.1 g of the EcoRV-Asp718fragment (0.9 kb) derived from the DNA fragments amplified by PCR and0.1 μg of the StuI-Asp718 fragment (9.06 kb) derived from the pAMoPRSAwere dissloved in 30 μl of T4 ligase buffer and ligated together with175 units of T4 DNA ligase at 12° C. for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated aspAMoPRSAL-35F, and its structure was confirmed by restriction enzymedigestion.

2. Secretory expression of cDNA encoding α2→3 sialyltransferase usingNamalwa KJM-1 cell as host

The plasmid pAMoPRSA (secretory expression vector; control) obtained inSec. 1(6) of this Example and the plasmid pAMoPRSAL-35F (plasmid forsecretory expression of α2→3 sialyltransferase) obtained in Sec. 1(7) ofthis Example were prepared using the plasmid preparationkit >plasmid<maxi kit (trade No. 41031; Qiagen). Each of the plasmidsthus obtained was ethanol precipitated and dissolved in TE buffer togive a concentration of 1 μg/μl. Then, both plasmids were independentlyintroduced into the Namalwa KJM-1 cells at a proportion of 4 μg per1×10⁶ cells by electroporation (Miyaji et al., Cytotechnology, 3, 133(1990)). The cells were suspended in 8 ml of RPMI1640.ITPSGF medium, andthe cells were cultured in a CO₂ incubator at 37° C. for 24 hours. Then,the cells were supplemented with G418 (GIBCO) to give a concentration of0.5 mg/ml and further cultured for 7 to 14 days to obtain transformants.Each of the transformants was suspended in 30 ml of RPMI1640.ITPSGFmedium containing 0.5 mg/ml of G418 at 1×10⁵ cells/ml, and the cellswere cultured in a CO₂ incubator at 37° C. for 8 days. Then,centrifugation at 160×g for 10 minutes gave the separation of asupernatant from the cells, and the supernatant was further centrifugedat 1500×g for 10 minutes. The culture supernatant thus obtained wasstored at -80° C. until it was used.

α2→3 Sialyltransferase encoded in the plasmid pAMoPRSAL-35F can readilybe purified using IgG Sepharose because of its secretory expression as afused protein with the IgG-binding region of Staphylococcus aureusprotein A. To the culture supernatant obtained above, sodium azide wasadded to give a final concentraion of 0.1%. Then, 100 μl of IgGSepharose (Pharmacia) which had been pre-treated according to theaccompanying instructions was added, and the mixture was gently stirredat 4° C. overnight. The IgG Sepharose was recovered by centrifugation at160×g for 10 minutes, and washed three times with 1 ml ofRPMI1640.ITPSGF medium. The sialyltranseferase activity was measured bydirectly using 5 μl of this IgG Sepharose. The activity measurement wascarried out by reacting in 30 μl of the assay solution (0.1M cacodylatebuffer (pH 6.5), 0.01M MnCl₂, 0.45% Triton X-100, 0.1 mM substrate, theabove IgG Sepharose (5 μl), 5 mM CMP-sialic acid (added or not added))at 37° C. for 2 hours, and then identifying the products by highperformance liquid chromatography (HPLC). As the substrate, varioussugar chains (lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT),lacto-N-fucopentaose III (LNFP-III) and lacto-N-fucopentaose V (LNFP-V);all are available from Oxford GlycoSystems, and the respectivestructures are shown in FIG. 1) fluorescence-labeled with aminopyridinewere used. The fluorescence labeling of the substrate was carried outaccording to the conventional method (Akihiro Kondo et al., Agric. Biol.Chem., 54, 2169 (1990)). The IgG Sepharose was allowed to react with anassay solution containing or not contaning CMP-sialic acid as a sugardonor. The reaction mixture was separated by HPLC, and the peaksappearing only with the assay solution containing CMP-sialic acid wereconsidered as the products. After completion of the reaction, the assaysolution was treated with at 100° C. for for 5 minutes, and centrifugedat 10,000×g for 10 minutes. The resulting supernatant was subjected toHPLC which was carried out on the TSKgel ODS-80T_(M) column (4.6 mm×30cm; Tosoh) eluting with 0.02M ammonium acetate buffer (pH 4.0) at atemperature of 50° C. at a rate of 1 ml/min. The products were detectedusing the fluorescence HPLC monitor model RF-535T (Shimazu Seisakusho)with an excitation wavelength of 320 nm and an emission wavelength of400 nm. The products was identified from the facts that the elution timewas coincided with that of the standard and that the substrate wasregenerated by sialidase treatment of the products. The quantitativeanalysis of the products was carried out with the use of pyridylaminatedlactose as the standard for comparison of fluorescence intensity. Theresults of this assay are shown in Table 1. The activity to varioussugar chain substrates is also shown in terms of a relative activitywhen the activity to the substrate LNnT is taken as 100.

                                      TABLE 1    __________________________________________________________________________                           α2→3 Sialyltransferase activity                                  pAMoPRSAL-35F                                            Known α2→3 ST                           pAMoPRSA   Relative                                            Relative    Substrate sugar chain  pmol   pmol                                      activity                                            activity    __________________________________________________________________________    Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LNnT)                           0      51.3                                      100    6     ##STR2##              0      8.1 16    Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNT)                           0      23.8                                      46    100     ##STR3##              0      28.8                                      56    __________________________________________________________________________

In cases where the IgG Sepharose derived from the culture supernatant ofNamalwa cells having the plasmid pAMoPRSAL-35F was used, α2→3sialyltransferase activity was detected when any of the sugar chains wasused as the substrate. On the other hand, in cases where the IgGSepharose derived from the culture supernatant of Namalwa cellscontaining the vector pAMoPRSA, no activity was detected when any of thesugar chains was used as the substrate. These results indicates thatα2→3 sialyltransferase undergo secretory production into the culturesupernatant as a fused protein with the IgG-binding region ofStaphylococcus aureus protein A and that it can readily be recovered andpurified using IgG Sepharose.

Also shown in Table 1 are the relative activities of a known α2→3sialyltransferase, for which purification has been reported so far(Wienstein et al., J. Biol. Chem., 257, 13845 (1982)), to various sugarchain substrates when its activity to the substrate LNT is taken as 100.The known α2→3 sialyltransferase exhibits a higher specificity to LNTthan LNnT, whereas the α2→3 sialyltransferase of the present inventionexhibits a higher specificity to LNnT than LNT. This indicates that theα2→3 sialyltransferase of the present invention is a novel enzyme havinga different substrate specificity from that of the known enzyme.

It was also found that the α2→3 sialyltransferase of the presentinvention can take LNFP-III as the substrate. No report has been made sofar on such α2→3 sialyltransferase having an enzymatic activity toLNFP-III. This indicates that the use of this enzyme can make possiblethe direct in vitro synthesis of sialyl-Le^(x) sugar chains from Le^(x)sugar chains.

Because the α2→3 sialyltransferase of the present invention can take notonly LNnT but also LNT as the substrate, it is possible to synthesizesialyl-Le^(x) sugar chains, as well as sialyl-Le^(a) sugar chains. Thatis, with the use of the α2→3 sialyltransferase of the present invention,the terminal structure of a sugar chain can be converted intoNeuAcα2-3Galβ1-4GlcNAc or NeuAcα2-3Galβ1-3GlcNAc, which are then madeinto a sialyl-Le^(x) sugar chain or a sialyl-Le^(a) sugar chain, usingα1→3 fucosyltransferase or α1→4 fucosyltransferase, respectively.Moreover, the α2→3 sialyltransferase of the present invention exhibits ahigher substrate specificity to LNnT than that attained by the knownα2→3 sialyltransferase, and is therefore superior to the known α2→3sialyltransferase with respect to the capability for synthesis ofsialyl-Le^(x) sugar chains.

EXAMPLE 4

Production by animal cells of α2→3 sialyltransferase derived from humanmelanoma cell strain WM266-4:

1. Cloning of α2→3 sialyltransferase cDNA (WM17) derived from humanmelanoma cell line WM266-4

(1) Isolation of mRNA from human melanoma WM266-4 cell line

From 1×10⁸ WM266-4 cells (ATCC CRL1676), about 30 μg of mRNA wasisolated using the mRNA extraction kit "Fast Track" (trade No. K1593-02;Invitrogen), according to the manufacturer's instructions accompanyingthe kit used.

(2) Preparation of cDNA library

From 8 μg of mRNA obatined above, double-stranded cDNA was preparedusing the cDNA synthesis kit "The Librarian I" (Invitrogen) with arandom primer as a primer. Then, each of the following SfiI linkers(Seq. ID: 5) as prepared in Example 1 was added, instead of BstXIlinker, at either terminus of the cDNA. The cDNA was fractioned in sizeby agarose gel electrophoresis and cDNA fragments larger than about 1.2kb were isolated.

SfiI Linkers:

    5'-CTTTAGAGCAC-3' (11 mer)

    3'-GAAATCTC-5' (8 mer)

Before use, these SfiI linkers (11 mer and 8 mer) had been independentlydissloved at 100 nM in 50 μl of T4 kinase buffer and phosphorylated with30 units of T4 polynucleotide kinase (Takara Shuzo) at 37° C. for 16hour. The specific reagents and procedures were as described in themanufacturer's instructions accompanying the kit used, except that theabove SfiI linkers were used in place of BstXI linkers. As the directexpression cloning vector, the vector pAMoPRC3Sc obtained in Sec. 1(5)of Example 3 was used.

First, 24 μg of the vector pAMoPRC3Sc was dissloved in 590 μl of Y-50buffer and digested with 80 units of SfiI at 37° C. for 16 hours. Then,the reaction mixture was taken at a volume of 5 μl for agarose gelelectrophoresis, and after the completion of digestion was confirmed,this vector was further digested with 40 units of BamHI at 37° C. for 2hours. The digestion with BamHI was done for the purpose of decreasingthe amount of background (i.e., clones containing no cDNA insert) at thetime of cDNA library construction. The reaction mixture was subjected toagarose gel electrophoresis to give an about 8.8 kb DNA fragment.

Then, 2 μg of the SfiI fragment (8.8 kb) derived from the pAMoPRC3Sc,together with the cDNA obtained above, was dissolved in 250 μl of T4ligase buffer and ligated together with 2000 units of T4 DNA ligase at12° C. for 16 hours. Thereafter, 5 μg of transfer RNA (tRNA) was added,and after ethanol precipitation, the resulting precipitate was disslovedin 20 μl of TE buffer. The reaction mixture was used to transformEscherichia coli LE392 strain (Maniatis et al., Molecular Cloning, 2.58,Cold Spring Harbor, 1989) by electroporation (William J. Dower et al.,Nucleic Acids Res., 16, 6127 (1988)) to give about 200,000 ampicillinresistant transformants.

(3) Cloning of α2→3 sialyltransferase cDNA (WM17)

About 200,000 ampicillin resistant transformants (cDNA library) obtainedin Sec. 1(2) of this Example were mixed, after which a plasmid wasprepared using >plasmid<maxi kit (trade No. 41031; Qiagen) which is aplasmid preparation kit. The obtained plasmid was ethanol precipitatedand the resulting precipitate was dissloved in TE buffer to give aconcentration of 1 μg/μl.

The above plasmid was introduced into the KJM-1 strain byelectroporation (Miyaji et al., Cytotechnology, 3, 133 (1990)) at aproportion of 4 μg per 1.6×10⁶ cells. After the introduction of plasmid,these cells were suspended in 8 ml of RPMI1640.ITPSGF medium, and thecells were cultured in a CO₂ incubator at 37° C. for 24 hours. Then, thecells were supplemented with G418 (GIBCO) to give a concentration of 0.5mg/ml and further cultured for 5 to 7 days to obtain transformants. Theobatined transformant was suspended in RPMI1640.ITPSGF medium containingRicinus communis 120 lectin (50 ng/ml) to give a concentration of 5×10⁴cells/ml, and the suspension was distributed in 200-μl portions intowells of a 96-well microtiter plate. The cells were cultured in a CO₂incubator at 37° C. for 4 weeks, and a certain strain was obtained whichhad become resistant to Ricinus communis 120 lectin. After culturing ofthis resistant cell, a plasmid was isolated from about 5×10⁶ cellsaccording to the Hirt method (Robert F. Margolskee et al., Mol. Cell.Biol., 8, 2837 (1988)). The isolated plasmid was introduced intoEscherichia coli strain LE392 by electroporation (William J. Dower etal., Nucleic Acids Res., 16, 6127 (1988)) to give an ampicillinresistant transformant. From this transformant, a plasmid was preparedusing >plasmid<maxi kit (Qiagen), and its structure was examined byrestriction enzyme digestion to find that it contained about 1.9 kbcDNA. The cDNA containing plasmid was designated as pAMoPRWM17. Whenthis plasmid was also introduced into the KJM-1 strain by the abovemethod, the transformant became resistant to Ricinus communis 120lectin; it was therefore found that this cDNA is a gene responsible forlectin resistance. The KJM-1 strain containing the plasmid DAMoPRWM17was able to grow even in the presence of 200 ng/ml Ricinus communes 220lectin.

2. Sequencing of α2→3 sialyltransferase cDNA (WM17)

(1) Incorporation of α2→3 sialyltransferase cDNA (WM17) into plasmidpUCl19 (see FIG. 27)

First, 1 μg of the plasmid pAMoPRWM17 was dissloved in 30 μl of Y-100buffer and digested with 20 units of EcoRV and 20 units of Asp718(Boehringer Mannheim) at 37° C. for 2 hours. The reaction mixture wassubjected to agarose gel electrophoresis to give an about 1.9 kb DNAfragment.

Separately, 1 μg of the plasmid pUCl19 (Messing et al., Methods inEnzymology, 153, 3 (1987)) was dissloved in 30 μl of K-20 buffer anddigested with 20 units of SmaI at 37° C. for 2 hours. Then, sodiumchloride was added to give an NaCl concentration of 100 mM, and thisplasmid was further digested with 20 units of Asp718 at 37° C. for 2hours. The reaction mixture was subjected to agarose gel electrophoresisto give an about 3.16 kb DNA fragment.

The DNA fragments thus obtained, i.e., 0.2 μg of the EcoRV-Asp718fragment (1.9 kb) derived from the pAMoPRWM17 and 0.1 μg of theSmaI-Asp718 fragment (3.16 kb) derived from the pUCl19 were dissolved in30 μl of T4 ligase buffer and ligated together with 175 units of T4 DNAligase at 12° C. for 16 hours. Escherichia coli HB101 strain wastransformed using this reaction mixture according to the method of Cohenet al. to obtain an ampicillin resistant strain. From this transformant,a plasmid was isolated according to the known method. This plasmid wasdesignated as pUCl19-WM17, and its structure was confirmed byrestriction enzyme digestion.

(2) Construction of deletion plasmids for sequencing

First, 2 μg of the plasmid PUCl19-WM17 was dissloved in 30 μl of Y-150buffer and digested with 20 units of BamHI and 20 units of SphI at 37°C. for 2 hours. After ethanol precipitation, the resuling precipitatewas dissloved in 100 μl of ExoIII buffer (accompanying the deletion kitfor kilo-sequence; Takara Shuzo). Independently, 2 μg of the sameplasmid was dissloved in 30 μl of Y-0 buffer and digested with 20 unitsof SacI at 37° C. for 2 hours. Then, sodium chloride was added to givean NaCl concentration of 150 mM and digested with 20 units of NotI at37° C. for 2 hours. After ethanol precipitation, the resultingprecipitate was dissloved in 100 μl of ExoIII buffer.

From the BamHI-SphI fragment derived from the plasmid pUCt19-WM17 andthe SacI-NotI fragment derived from the same plasmid, several tens ofdeletion plasmids were respectively prepared using the deletion kit forkilo-sequence (Takara Shuzo). Specific reagents and procedures were asdescribed in the manufacturer's instructions accompanying the kit used.

The nucleotide sequence of the deletion plasmid obtained above wasdetermined using the Taq DyeDeoxy terminator cycle sequencing kit (tradeNo. 401113; Applied Biosystems). The determined nucleotide sequence isshown in the sequence listing (Seq. ID: 6). A poly A tail follows 1742base pairs (bp) nucleotide. From this nucleotide sequence, it was foundthat this gene encodes a protein consisting of 329 amino acids. Theamino acid sequence of this protein is also shown in the sequencelisting (Seq. ID: 7). It was also found that the amino acid sequence hasabout 91% homology with that encoded by α2→3 sialyltransferase cDNAcloned from TYH cells in Example 4. From these results, it is consideredthat the gene (WM17) isolated in terms of resistance to Ricinus communis120 lectin encodes α2→3 sialyltransferase.

3. Measurement of α2→3 sialyltransferage activity of KJM-1 straincontaining WM17 expression plasmid

In the same manner as described in Sec. 5 of Example 5, comparison ofα2→3 sialyltransferase activity was made between the KJM-1 straincontaining the WM17 expression plasmid (pAMoPRWM17) and the KJM-1 straincontaining the plasmid pAMoPRC3Sc as a control. As the result, the α2→3sialyltransferase activity of the KJM-1 strain containing the plasmidpAMoPRWM17 was 6 to 7 times higher than that of the KJM-1 straincontaining the plasmid pAMoPRC3Sc.

4. Synthesis of sialyl-Le^(x) sugar chain in KJM-1 strain havingexpression plasmid for α2→3 sialyltransferase

The KJM-1 strain containing the pAMoPRWM17 (expression plasmid for α2→3sialyltransferase) obatined in Sec. 1 of Example 4 and the KJM-1 straincontaining the pAMoPRC3Sc (control plasmid) were independently culturedin the RPMI1640.ITPSGF medium containing 0.5 mg/ml of G418, after whichabout 1×10⁶. cells of each strain were taken in a microtube (1.5 ml;Eppendorf) and collected by centrifugation at 550×g for 7 minutes. Then,these cells were washed with 1 ml of A-PBS and subjected to indirectfluorescent antibody staining with KM93 (Shitara et al., AnticancerRes., 9, 999 (1989)) which is an antibody reacting with thesialyl-Le^(x) sugar chain, thereby examining the production ofsialyl-Le^(x) sugar chain in these cells. The collected cells of eachstrain were suspended in 50 μl (10 μg/ml) of KM93 and allowed to reactat 4° C. for 1 hour. Then, these cells were washed three times withA-PBS and suspended in 20 μl of fluorescence-labeled anti-mouse IgG andIgM antibodies (Cappel, used after 20-fold dilution with A-PBS),followed by reaction at 4° C. for 30 minutes. After washing three timeswith A-PBS, these cells were again suspended in A-PBS and subjected toanlysis using EPICS elite flow cytometer (Coulter).

As a control, the same analysis was carried out using the normal mouseserum diluted 500-fold with A-PBS in place of KM93.

The results are shown in FIG. 28. It is found that for the KJM-1 straincontaining the direct expression cloning vector pAMoPRC3Sc (controlplasmid), the cells stained with KM93 exhibit a higher fluorescenceintensity than that of the control. This indicates that the KJM-1 strainis originally able to express the sialy-Le^(x) sugar chains. Also foundis that the fluorescence intensity obtained when the cells of the KJM-1strain containing the pAMoPRWM17 (α2→3 sialyltransferase expressionplasmid) were stained with KM93 is further higher than that obatinedwhen the cells of the KJM-1 strain having the pAMoPRC3Sc (controlplasmid) were stained with KM93. This indicates that α2→3sialyltransferase encoded in the gene WM17 can synthesize sialyl-Le^(x)sugar chains in cells.

5. Secretory production by animal cells of α2→3 sialyltransferasederived from WM266-4 cell line

(1) Construction of plasmid pAMoPRSAW17-31F for secretory expression ofcDNA encoding α2→3 sialyltransferase (see FIG. 29)

In view of the amino acid sequence, α2→3 sialyltransferase encoded inthe cloned gene WM17 seems to have a structure where 8 amino acids inthe N-terminal portion are put out at the cytoplasma side, the highlyhydrophobic region consisting of the subsequent 18 amino acids is usedfor binding the membrane, and most of the remaining C-terminal portion(including the catalytic site) is exposed to the internal cavity ofGolgi's apparatus. According to the method as described below,therefore, the secretory production of α2→3 sialyltransferase wasachieved by removing the membrane-binding region from α2→3sialyltransferase and-adding-instead the signal sequence of granulocytecolony stimulating factor and the IgG-binding region of Staphylococcusaureus protein A. The gene portion encoding a certain region followingthe membrane-binding region of α2→3 sialyltransferase (from 31st Phe to329th Phe) was prepared by PCR method and inserted into the secretoryexpression vector pAMoPRSA obtained in Sec. 1(6) of Example 3.

As a set of primers using PCR, the following two synthetic DNAfragments, i.e., W17-A(31F) (44 mer) and W17-C (36 mer), weresynthesized by DNA synthesizer model 380A (Applied Biosystems).

    W17-A(31F) (44 mer): 5'-CTCTCCGATATCTGTTTTATTTTCCCATCCCAGAGAAGAAGGAG-3'

    W17-C (36 mer) 5'-GATTAAGGTACCAGGTCAGAAGGACGTGAGGTTCTT-3'

The primers using PCR W17-A (31F) and W17-C are designed to have anEcoRV site and an Asp718 site, respectively, so that DNA fragmentsamplified by PCR can be incorporated between the S tuI site and theAsp718 site of the plasmid pAMoPRSA after digestion with EcoRV andAsp718. The PCR was carried out using a GeneAmp™ DNA amplificationreagent kit with AmpliTaq™ recombinant Taq DNA polymerase (TakaraShuzo). The reaction mixture was prepared according to the method asdescribed in the kit, and Perkin Elmer Cetus DNA thermal cycler (TakaraShuzo) was used for incubation. Thirty cycles of amplification werecarried out according to the following scheme: 94° C. for 1 minute, 55°C. for 1 minute and 72° C. for 3 minutes Then, further incubation wascarried out at 72° C. for 7 minutes. As a templete, 1 ng of the plasmidpUCl19-WM17 obtained in Sec. 2(1) of this Example was used. Aftercompletion of the reaction, chloroform extraction and ethanolprecipitation were successively carried out. The resulting precipitatewas then dissloved in 30 μl of Y-100 buffer and digested with 20 unitsof EcoRV and 20 units of Asp718 at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis to give an about0.91 kb DNA fragment.

Separately, 2 μg of the plasmid pAMoPRSA was dissloved in 30 μl of Y-100buffer and digested with 20 units of StuI and 20 units of Asp718 at 37°C. for 2 hours. The reaction mixture was subjected to agarose gelelectrophoresis to give an about 9.06 kb DNA fragment.

Then, 0.1 μg of the EcoRV-ASp718 fragment (0.91 kb) derived from theamplified DNA by PCR and 0.1 μg of the StuI-ASp718 fragment (9.06 kb)derived from the plasmid pAMoPRSA were dissloved in 30 μl of T4 ligasebuffer and ligated together with 175 units of T4 DNA ligase at 12° C.for 16 hours.

Escherichia coli HB101 strain was transformed using this reactionmixture according to the method of Cohen et al. to obtain an ampicillinresistant strain. From this transformant, a plasmid was isolatedaccording to the known method. This plasmid was designated aspAMoPRSAW17-31F, and its structure was confirmed by restriction enzymedigestion.

(2) Secretory expression of cDNA encoding α2→3 sialyltransferase withNamalwa KJM-1 cells used as a host

The plasmid pAMoPRSA (secretory expression vector; control) obtained inSec. 1(6) of Example 3 and the plasmid pAMoPRSAW17-31F (secretoryexpression plasmid for α2→3 sialyltransferase) obtained in Sec. 5' (1)of this Example were prepared using the plasmid preparationkit, >plasmid<maxi kit (trade No. 41031; Qiagen). Each of the plasmidsthus obtained was precipitated by ethanol and dissloved in TE buffer togive a concentration of 1μg/μl. Then, both plasmids were independentlyintroduced into the Namalwa KJM-1 cells at a proportion of 4 μg per1.6×10⁶ cells by electroporation (Miyaji et al., Cytotechnology, 3, 133(1990)). The cells were suspended in 8 ml of RPMI1640.ITPSGF medium, andthe cells were cultured in a CO₂ incubator at 37° C. for 24 hours. Then,the cells were supplemented with G418 (GIBCO) at a concentration of 0.5mg/ml and further cultured for 7 to 14 days, resulting in transformants.Each of the transformants was suspended in 30 ml of RPMI1640.ITPSGFmedium containing 0.5 mg/ml of G418 to give a concentration of 1×10⁵cells/ml, and the cells were cultured in a CO₂ incubator at 37° C. for 8days. Then, centrifugation at 160×g for 10 minutes gave the separationof a supernatant from the cells, and the supernatant was furthercentrifuged-at 1500×g for 10 minutes. The culture supernatant thusobtained was stored at -80° C. until it was used.

α2→3 sialyltransferase encoded in the plasmid pAMoPRSAW17-31F canreadily be purified using IgG Sepharose because of its secretoryproduction as a fused protein with the IgG-binding region ofStaphylococcus aureus protein A. To the culture supernatant obtainedabove, sodium azide was added to give a final concentration of 0.1%.Then, 100 μl of IgG Sepharose (Pharmacia) which had been pre-treatedaccording to the accompanying instructions was added, and the mixturewas gently stirred at 4° C. overnight. The IgG Sepharose was recoveredby centrifugation at 160×g for 10 minutes, and washed three times with 1ml of RPMI1640.ITPSGF medium. The sialyltransferase activity wasmeasured by directly using 5 μl of this IgG Sepharose. The activitymeasurement was carried out by reacting in 30 μl of the assay solution(0.1M cacodylate buffer (pH 6.5), 0.01M MnCl₂, 0.45% Triton X-100, 0.1mM substrate, the above IgG Sepharose (5 μl), 5 mM CMP-sialic acid(added or not added)) at 37° C. for 2 hours, and then identifying theproducts by high performance liquid chromatography (HPLC). As thesubstrate, various sugar chains (lacto-N-neotetraose (LNnT),lacto-N-tetraose (LNT), lacto-N-fucopentaose III (LNFP-III) andlacto-N-fucopentaose V (LNFP-V); all are available from OxfordGlycoSystems, and the respective structures are shown in FIG. 1)fluorescence-labeled with aminopyridine were used. The fluorescencelabeling of the substrate was carried out according to the conventionalmethod (Akihiro Kondo et al., Agric. Biol. Chem., 54, 2169 (1990)). TheIgG Sepharose was allowed to react with an assay solution containing ornot containing CMP-sialic acid as a sugar donor. The reaction mixturewas analysed by HPLC, and the peaks appearing only with the assaysolution containing CMP-sialic acid were considered as the products.After completion of the reaction, the assay solution was treated at 100°C. for 5 minutes, and centrifuged at 10,000×g for 10 minutes. Then, 10μl of the resulting supernatant was subjected to HPLC which was carriedout on the TSKgel ODS-80T_(M) column (4.6 mm×30 cm; Tosoh) eluting with0.02M ammonium acetate buffer (pH 4.0) at a temperature of 50° C. at arate of 1 ml/min. The products were detected using the fluorescence HPLCmonitor model RF-535T (Shimazu Seisakusho) with an excitation wavelengthof 320 nm and an emission wavelength of 400 nm. The products wereidentified from the facts that the elution time was coincided with thatof the standard and that the substrate was regenerated by sialidasetreatment of the products. The quantitative analysis of the products wascarried out with the use of pyridylaminated lactose as the standard forcomparison of fluorescence intensity. The results of this assay areshown in Table 2. The activity to various sugar chain substrates is alsoshown in terms of a relative activity when the activity to the substrateLNnT is taken as 100.

                                      TABLE 2    __________________________________________________________________________                           α2→3 Sialyltransferase activity                                  pAMoPRSAW17-31F                                             Known α2→3 ST                           pAMoPRSA    Relative                                             Relative    Substrate sugar chain  pmol   pmol activity                                             activity    __________________________________________________________________________    Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LNnT)                           0      113.8                                       100    6     ##STR4##              0      9.4   8    Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNT)                           0      47.5 42    100     ##STR5##              0      36.3 32    __________________________________________________________________________

In cases where the IgG Sepharose derived from the culture supernatant ofNamalwa cells having the plasmid pAMoPRSAW17-31F was used, α2→3sialyltransferase activity was detected when any of the sugar chains wasused as the substrate. On the other hand, in cases where the IgGSepharose derived from the culture supernatant of Namalwa cells havingthe vector pAMoPRSA, no activity was detected when any of the sugarchains was used as the substrate. These results indicate that α2→3sialyltransferase undergo secretory production into the culturesuprnatant as a fused protein with the IgG-binding region ofStaphylococcus aureus protein A and that it can readily be recovered andpurified using IgG Sepharose.

Also shown in Table 2 are the relative activities of a known α2→3sialyltransferase, for which purification has been reported so far(Wienstein et al., J. Biol. Chem., 257, 13845 (1982)), to various sugarchain substrates when its activity to the substrate LNT is taken as 100.The known α2→3 sialyltransferase exhibits a higher specificity to LNTthan LNnT, whereas the α2→3 sialyltransferase of the present inventionexhibits a higher specificity to LNnT than LNT. This indicates that theα2→3 sialyltransferase of the present invention is a novel enzyme havinga different susbtrate specificity from that of the known enzyme.

It was also shown that the α2→3 sialyltransferase of the presentinvention can take LNFP-III as the substrate. No report has been made sofar on such α2→3 sialyltransferase having an enzymatic activity toLNFP-III. This indicates that the use of this enzyme can make possiblethe direct in vitro synthesis of sialyl-Le^(x) sugar chains from Le^(x)sugar chains.

Because the α2→3 sialyltransferase of the present invention can take notonly LNnT but also LNT as the substrate, it is possible to synthesizesialyl-Le^(a) sugar chains, as well as sialyl-Le^(x) sugar chains. Thatis, with the use of the α2→3 sialyltransferase of the present invention,the terminal structure of a sugar chain can be converted intoNeuAcα2-3Galβ1-4GlcNAc or NeuAcα2-3Galβ1-3GlcNAc, which are then madeinto a sialyl-Le^(x) sugar chain or a sialyl-Le^(a) sugar chain, usingα1→3 fucosyltransferase or α1→4 fucosyltransferase, respectively.Moreover, the α2→3 sialyltransferase of the present invention exhibits ahigher substrate specificity to LNnT than that attained by the knownα2→3 sialyltransferase, and is therefore supeior to the known α2→3sialyltransferase with respect to the capability for synthesis ofsialyl-Le^(x) sugar chains.

    __________________________________________________________________________       SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii ) NUMBER OF SEQUENCES: 7    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1919     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii ) ORIGINAL SOURCE:     (A) ORGANISM: human     (B) CELL LINE: TYH cell     (C) CELL TYPE: histiocytoma cell    (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    GTCGGGG CGCCTAAC GGATCGGA GCTGCGCG CGGATTTA CCTCGCC CCGCCCCG CTCCAC 60    CCGCGAG GGTGGCCC GAGGCAGC CGGGATGA CACTTCTC CCCAGGAA CCCTGCT ATCTGC 120    TGAGAAA CATGACC AGCAAA TCTCACT GGAAGCT CCTGGCC CTGGCT CTG17 0     MetThr SerLys SerHis TrpLys LeuLeu AlaLeu AlaLeu     1  5  10    GTCCTT GTTGTT GTCATG GTGTGGT ATTCCA TCTCCCG AGAAGAT AGG 218    ValLeu ValVal ValMet ValTrp TyrSer IleSer ArgGlu AspArg    15  20  25   30    TACATT GAGTTC TTTTAT TTTCCC ATCTCAG AGAAGAA AGAGCCA TGC 266    TyrIle GluPhe PheTyr PhePro IleSer GluLys LysGlu ProCys      35  40  45    TTCCAG GGTGAG GCAGAGA GACAGGC CTCTAAG ATTTTT GGCAACC GT31 4    PheGln GlyGlu AlaGlu ArgGln AlaSer LysIle PheGly AsnArg     50  5 5  60    TCTAGG GACCAG CCCATC TTTCTGC AGCTTAA GGATTAT TTCTGG GTA3 62    SerArg AspGln ProIle PheLeu GlnLeu LysAsp TyrPhe TrpVal     65  70  75    AAGACG CCATCC ACCTATG AGCTGCC CTTTGGG ACTAAA GGAAGTG AA41 0    LysThr ProSer ThrTyr GluLeu ProPhe GlyThr LysGly SerGlu    80   85  90    GACCTT CTTCTC CGGGTGC TGGCCAT CACTAGC TATTCT ATACCTG AG45 8    AspLeu LeuLeu ArgVal LeuAla IleThr SerTyr SerIle ProGlu    95  100  105  1 10    AGCATA AAGAGC CTGGAGT GTCGTCG CTGTGT TGTGGT GGGAAAT GGG 506    SerIle LysSer LeuGlu CysArg ArgCys ValVal ValGly AsnGly      115  120  125    CACCGG TTGCGG AACAGCT CGCTGGG CGGTGTC ATCAAC AAGTACG AC55 4    HisArg LeuArg AsnSer SerLeu GlyGly ValIle AsnLys TyrAsp     130  13 5  140    GTGGTC ATCAGA TTGAACA ATGCTCC TGTGGCT GGCTAC GAGGGAG AT60 2    ValVal IleArg LeuAsn AsnAla ProVal AlaGly TyrGlu GlyAsp     145  150  155    GTGGGC TCCAAG ACCACCA TACGTCT CTTCTA TCCTGAG TCGGCC CAC6 50    ValGly SerLys ThrThr IleArg LeuPhe TyrPro GluSer AlaHis    160  1 65  170    TTTGAC CCTAAA ATAGAAA ACAACCC AGACACG CTCTTG GTCCTG GTA6 98    PheAsp ProLys IleGlu AsnAsn ProAsp ThrLeu LeuVal LeuVal    175  180  185  1 90    GCTTTC AAGGCG ATGGACT TCCACTG GATTGAG ACCATC TTGAGTG AT74 6    AlaPhe LysAla MetAsp PheHis TrpIle GluThr IleLeu SerAsp      195  200  205    AAGAAG CGGGTG CGAAAAG GCTTCTG GAAACAG CCTCCC CTCATC TGG7 94    LysLys ArgVal ArgLys GlyPhe TrpLys GlnPro ProLeu IleTrp     210  21 5  220    GATGTC AACCCC AAACAGG TCCGGAT TCTAAAC CCCTTC TTTATGG AG84 2    AspVal AsnPro LysGln ValArg IleLeu AsnPro PhePhe MetGlu     225  230  235    ATTGCA GCAGAC AAGCTCC TGAGCCT GCCCAT ACAACAG CCTCGA AAG8 90    IleAla AlaAsp LysLeu LeuSer LeuPro IleGln GlnPro ArgLys    240  2 45  250    ATCAAG CAGAAG CCAACCA CGGGTCT GCTAGCC ATCACC TTGGCTC TA93 8    IleLys GlnLys ProThr ThrGly LeuLeu AlaIle ThrLeu AlaLeu    255  260  265  2 70    CACCTC TGCGAC TTAGTGC ACATTGC TGGCTTT GGCTAT CCAGATG CC98 6    HisLeu CysAsp LeuVal HisIle AlaGly PheGly TyrPro AspAla      275  280  285    TCCAAC AAGAAG CAGACCA TCCACTA CTATGAA CAGATC ACACTTA AG103 4    SerAsn LysLys GlnThr IleHis TyrTyr GluGln IleThr LeuLys     290  29 5  300    TCTATG GCGGGA TCAGGCC ATAATGT CTCCCAA GAGGCT ATCGCCA TC108 2    SerMet AlaGly SerGly HisAsn ValSer GlnGlu AlaIle AlaIle     305  310  315    AAGCGG ATGCTA GAGATGG GAGCTGT CAAGAAC CTCACA TACTTCT GA113 0    LysArg MetLeu GluMet GlyAla ValLys AsnLeu ThrTyr PheTER    320  3 25  330    CTTGGAT GGGAGCTG TAACACCT TGGTTCC CTACTTTG CCATCTG AGTAGGCC CTGTCTA 1190    CAGCTTA GGGGTTC CTGGTGC CAGTACAAT CCAATTGA ACTCACCC TCAATGGA GAGGGT1 250    GTTCTGG GGCTGTCC AGGTCTCC AGAGAGGC TATGTCCC TGCCTAC TTTGGTG GATTTCA 1310    AATCCAG ACAGGGTA GTCACACC AGGCTACA GGAGCCTGG CTAAAAGG GGGGGGGG GGCT137 0    GTTACT GTGGCAT CCCCTCT CTCAGCCAG CACAAAGA GCTGTTT TGTTTTG TTTTGTTT T1430    GTTTTG TTTTGTT TCGTTTT GTTTCGTT TTGTTTT GTCTTGT ATTGTTGG TGGTGGT TGG1490    TTGGGTT TTGTTTG TTTGTTT TTGTTTGC TTTGTTT TGTTCTT GAGACAGG GTCTGACT G1550    TGAAACC CTGGCTA ATCTGGAA CTCACTAT GTAGACCA GACTGGT CTTGAAC TCACAGAG 1610    ATCCAAC TGCCTTT GCCTCCCA AGTGTTGG GATGAAAG GCATGTA CTACGCC TGGCCCCA 1670    ACACCAA GAGATTA TTTAACA TTCTATTT AATTAAGG GGTAGGAA AATGAATG GGCTGGT 1730    CCCAGGA TGTTCAT GAAAGGGA CACAATAC AGTGTTCT GCCCACTT TTTAATA AAATTTA 1790    CATGTGA TTGGCCT GTTAAGGC CCAATTCT AGAGCTGG CCTCCCAG AAAGATGG AGGCAT1 850    CAAGAGT GGGAGGGT GTCCTCC AGAGAGGG GTTGCTAC TTCCCAGC AGGCATGG GGGGAG1 910    CATTGAC AA      191 9    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 333     (B) TYPE: amino acid     (C) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii ) ORIGINAL SOURCE:     (A) ORGANISM: human     (B) CELL LINE: TYH cell     (C) CELL TYPE: histiocytoma cell    (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    MetThr SerLys SerHis TrpLys LeuLeu AlaLeu AlaLeu ValLeu    1  5  10  15    ValVal ValMet ValTrp TyrSer IleSer ArgGlu AspArg TyrIle     20  2 5  30    GluPhe PheTyr PhePro IleSer GluLys LysGlu ProCys PheGln     35  40  45    GlyGlu AlaGlu ArgGln AlaSer LysIle PheGly AsnArg SerArg    50   55  60    AspGln ProIle PheLeu GlnLeu LysAsp TyrPhe TrpVal LysThr    65  70  75   80    ProSer ThrTyr GluLeu ProPhe GlyThr LysGly SerGlu AspLeu      85  90  95    LeuLeu ArgVal LeuAla IleThr SerTyr SerIle ProGlu SerIle     100  10 5  110    LysSer LeuGlu CysArg ArgCys ValVal ValGly AsnGly HisArg     115  120  125    LeuArg AsnSer SerLeu GlyGly ValIle AsnLys TyrAsp ValVal    130  1 35  140    IleArg LeuAsn AsnAla ProVal AlaGly TyrGlu GlyAsp ValGly    145  150  155  1 60    SerLys ThrThr IleArg LeuPhe TyrPro GluSer AlaHis PheAsp      165  170  175    ProLys IleGlu AsnAsn ProAsp ThrLeu LeuVal LeuVal AlaPhe     180  18 5  190    LysAla MetAsp PheHis TrpIle GluThr IleLeu SerAsp LysLys     195  200  205    ArgVal ArgLys GlyPhe TrpLys GlnPro ProLeu IleTrp AspVal    210  2 15  220    AsnPro LysGln ValArg IleLeu AsnPro PhePhe MetGlu IleAla    225  230  235  2 40    AlaAsp LysLeu LeuSer LeuPro IleGln GlnPro ArgLys IleLys      245  250  255    GlnLys ProThr ThrGly LeuLeu AlaIle ThrLeu AlaLeu HisLeu     260  26 5  270    CysAsp LeuVal HisIle AlaGly PheGly TyrPro AspAla SerAsn     275  280  285    LysLys GlnThr IleHis TyrTyr GluGln IleThr LeuLys SerMet    290  2 95  300    AlaGly SerGly HisAsn ValSer GlnGlu AlaIle AlaIle LysArg    305  310  315  3 20    MetLeu GluMet GlyAla ValLys AsnLeu ThrTyr Phe      325  330    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 52     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid synthetic DNA    (iii ) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    TCGACAA GCTTGAT ATCGGCCT GTGAGGCC TCACTGGCC GCGGCCGC GGTAC 5 2    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 44     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid synthetic DNA    (iii ) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    CGCGGC CGCGGCCA GTGAGGCC TCACAGGC CGATATCA AGCTTG  4 4    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 11     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid synthetic DNA    (iii ) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CTTTAGA GCAC      11    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1766     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii ) ORIGINAL SOURCE:     (A) ORGANISM: human     (B) CELL LINE: WM266-4 cell     (C) CELL TYPE: melanoma    (iv) SEQUENCE DESCRIPTION:SEQ ID NO:6:    CGGTCAG GTCCAGCA CTTGGGAG CTGACTG TGCTGGAGG TGACAGGC TTTGCGGG GTCCG 60    CCTGTG TGCAGGAG TCGCAAGG TCGCTGAG CAGGACCCA AAGGTGGC CCGAGGCA GCCGG1 20    GATGACA GCTCTCC CCAGGAAT CCTGCTGC CTGCTGAG AAACATG GTCAGC AAG 174         MetV alSerL ys         1    TCCCGC TGGAAG CTCCTGG CCATGT TGGCTCT GGTCCT GGTCGT CATG 222    SerArg TrpLys LeuLeu AlaMet LeuAla LeuVal LeuVal ValMet    5  10  15   20    GTGTGG TATTCC ATCTCCC GGGAAGA CAGTTTT TATTTT CCCATC CCA2 70    ValTrp TyrSer IleSer ArgGlu AspSer PheTyr PhePro IlePro      25  30  35    GAGAAG AAGGAG CCGTGCC TCCAGGG TGAGGCA GAGAGC AAGGCCT CT31 8    GluLys LysGlu ProCys LeuGln GlyGlu AlaGlu SerLys AlaSer     40  4 5  50    AAGCTC TTTGGC AACTACT CCCGGGA TCAGCCC ATCTTC CTGCGGC TT36 6    LysLeu PheGly AsnTyr SerArg AspGln ProIle PheLeu ArgLeu     55  60  65    GAGGAT TATTTC TGGGTCA AGACGCC ATCTGCT TACGAG CTGCCC TAT4 14    GluAsp TyrPhe TrpVal LysThr ProSer AlaTyr GluLeu ProTyr    70   75  80    GGGACC AAGGGG AGTGAGG ATCTGC TCCTCCG GGTGCT AGCCATC ACC 462    GlyThr LysGly SerGlu AspLeu LeuLeu ArgVal LeuAla IleThr    85  90  95  1 00    AGCTCC TCCATC CCCAAGA ACATCCA GAGCCTC AGGTGC CGCCGCT GT51 0    SerSer SerIle ProLys AsnIle GlnSer LeuArg CysArg ArgCys      105  110  115    GTGGTC GTGGGG AACGGGC ACCGGCT GCGGAAC AGCTCA CTGGGAG AT55 8    ValVal ValGly AsnGly HisArg LeuArg AsnSer SerLeu GlyAsp     120  12 5  130    GCCATC AACAAG TACGATG TGGTCA TCAGATT GAACAAT GCCCCA GTG6 06    AlaIle AsnLys TyrAsp ValVal IleArg LeuAsn AsnAla ProVal     135  140  145    GCTGGC TATGAG GGTGACG TGGGCTC CAAGACC ACCATG CGTCTCT TC65 4    AlaGly TyrGlu GlyAsp ValGly SerLys ThrThr MetArg LeuPhe    150  1 55  160    TACCCT GAATCT GCCCACT TCGACCC CAAAGTA GAAAAC AACCCAG AC70 2    TyrPro GluSer AlaHis PheAsp ProLys ValGlu AsnAsn ProAsp    165  170  175  1 80    ACACTC CTCGTC CTGGTAG CTTTCAA GGCAATG GACTTC CACTGGA TT75 0    ThrLeu LeuVal LeuVal AlaPhe LysAla MetAsp PheHis TrpIle      185  190  195    GAGACC ATCCTG AGTGATA AGAAGCG GGTGCGA AAGGGT TTCTGGA AA79 8    GluThr IleLeu SerAsp LysLys ArgVal ArgLys GlyPhe TrpLys     200  20 5  210    CAGCCT CCCCTC ATCTGGG ATGTCAA TCCTAAA CAGATT CGGATTC TC84 6    GlnPro ProLeu IleTrp AspVal AsnPro LysGln IleArg IleLeu     215  220  225    AACCCC TTCTTC ATGGAGA TTGCAGC TGACAAA CTGCTG AGCCTGC CA89 4    AsnPro PhePhe MetGlu IleAla AlaAsp LysLeu LeuSer LeuPro    230  2 35  240    ATGCAA CAGCCA CGGAAGA TTAAGCA GAAGCCC ACCACG GGCCTGT TG94 2    MetGln GlnPro ArgLys IleLys GlnLys ProThr ThrGly LeuLeu    245  250  250  2 60    GCCATC ACGCTG GCCCTCC ACCTCTG TGACTTG GTGCAC ATTGCCG GC99 0    AlaIle ThrLeu AlaLeu HisLeu CysAsp LeuVal HisIle AlaGly      265  270  275    TTTGGC TACCCA GACGCCT ACAACAA GAAGCAG ACCATT CACTACT AT103 8    PheGly TyrPro AspAla TyrAsn LysLys GlnThr IleHis TyrTyr     280  28 5  290    GAGCAG ATCACG CTCAAGT CCATGGC GGGGTCA GGCCAT AATGTCT CC108 6    GluGln IleThr LeuLys SerMet AlaGly SerGly HisAsn ValSer     295  300  305    CAAGAG GCCCTG GCCATTA AGCGGAT GCTGGAG ATGGGA GCTATCA AG113 4    GlnGlu AlaLeu AlaIle LysArg MetLeu GluMet GlyAla IleLys    310  3 15  320    AACCTC ACGTCC TGCTGAC CTGGGCAAG AGCTGTAG CCTGTCG GTTGC 11 82    AsnLeu ThrSer PheTER    325  329    CTACTC TGCTGTC TGGGTGAC CCCCATGC GTGGCTG TGGGGGTG GCTGGTGC CAGTATGA 1242    CCCACTT GGACTCAC CCCCTCT TGGGGAGG GAGTTCTG GGCCTGGC CAGGTCTG AGATGA1 302    GGCCATG CCCCTGG CTGCTCT TATGGAGC CGAGATCC AGTCAGGG TGGGGGCG CTGGAGC 1362    AAGAGAT TATTTAA TGGGCTAT TTAATTAA GGGGTAGG AAGGTGCT GTGGGCTG GTCCCA 1482    CACATCC AGGAAAGA GGCCAGTA GAGAATTC TGCCCACT TTTTATA AAAACTTA CAGCGA1 542    TGGCCCC ACCAAGGC CTAGACAC GGCACTGG CCTCCCAG GAGGGCAG GGGCATTG GGAAT16 02    GGGTGGG TGCCCTC CAGAGAGG GGCTGCTA CCTCCCAG CAGGCATG GGAAGAGC ACTGGT1 662    ACTATT TTTCCTA AAACGGAA AAAAAAAAA AAAAAAAA AAAAAA  1 766    (2) INFORMATION FOR SEQ ID NO: 7:     (A) LENGTH: 329     (B) TYPE: amino acid     (C) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii ) ORIGINAL SOURCE:     (A) ORGANISM: human     (B) CELL LINE: WM266-4 cell     (C) CELL TYPE: melanoma    (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    MetVal SerLys SerArg TrpLys LeuLeu AlaMet LeuAla LeuVal    1  5  10  15    LeuVal ValMet ValTrp TyrSer IleSer ArgGlu AspSer PheTyr     20  2 5  30    PhePro IlePro GluLys LysGlu ProCys LeuGln GlyGlu AlaGlu     35  40  45    SerLys AlaSer LysLeu PheGly AsnTyr SerArg AspGln ProIle    50   55  60    PheLeu ArgLeu GluAsp TyrPhe TrpVal LysThr ProSer AlaTyr    65  70  75   80    GluLeu ProTyr GlyThr LysGly SerGlu AspLeu LeuLeu ArgVal      85  90  95    LeuAla IleThr SerSer SerIle ProLys AsnIle GlnSer LeuArg     100  10 5  110    CysArg ArgCys ValVal ValGly AsnGly HisArg LeuArg AsnSer     115  120  125    SerLeu GlyAsp AlaIle AsnLys TyrAsp ValVal IleArg LeuAsn    130  1 35  140    AsnAla ProVal AlaGly TyrGlu GlyAsp ValGly SerLys ThrThr    145  150  155  1 60    MetArg LeuPhe TyrPro GluSer AlaHis PheAsp ProLys ValGlu      165  170  175    AsnAsn ProAsp ThrLeu LeuVal LeuVal AlaPhe LysAla MetAsp     180  18 5  190    PheHis TrpIle GluThr IleLeu SerAsp LysLys ArgVal ArgLys     195  200  205    GlyPhe TrpLys GlnPro ProLeu IleTrp AspVal AsnPro LysGln    210  2 15  220    IleArg IleLeu AsnPro PhePhe MetGlu IleAla AlaAsp LysLeu    225  230  235  2 40    LeuSer LeuPro MetGln GlnPro ArgLys IleLys GlnLys ProThr      245  250  255    ThrGly LeuLeu AlaIle ThrLeu AlaLeu HisLeu CysAsp LeuVal     260  26 5  270    HisIle AlaGly PheGly TyrPro AspAla TyrAsn LysLys GlnThr     275  280  285    IleHis TyrTyr GluGln IleThr LeuLys SerMet AlaGly SerGly    290  2 95  300    HisAsn ValSer GlnGlu AlaLeu AlaIle LysArg MetLeu GluMet    305  310  315  3 20    GlyAla IleLys AsnLeu ThrSer Phe      325 32 9

What is claimed is:
 1. A cDNA encoding an α2→3 sialyltransferase havingan amino acid sequence as designated by Seq. ID: 2 or 7 or a DNA havinghomology with said cDNA.
 2. A cDNA having a nucleotide sequence asdesignated by Seq. ID: 1 or
 6. 3. A recombinant vector containing a cDNAwhich encodes α2→3 sialyltransferase having an amino acid sequence asdesignated by Seq. ID: 2 or
 7. 4. A recombinant vector containing a cDNAwhich encodes an amino acid sequence as designated by Seq. ID: 1 or 6.5. A process of preparing a cDNA according to claim 1 or 2, whichcomprises the steps of:constructing a cDNA library by incorporating intoa vector, a cDNA synthesized using as an template an mRNA extracted froman animal cell; introducing said cDNA library into a cell and preparinga culture of the obtained cell in the presence of a lectin having anactivity to inhibit the growth of said cell; and separating the growncell from said culture and isolating from said cell, a DNA which encodesα2→3 sialyltransferase.
 6. A process of preparing a recombinant vectoraccording to claim 3 or 4, which comprises the steps of:constructing acDNA library by incorporating into a vector, a cDNA synthesized using asan template an mRNA extracted from an animal cell; introducing said cDNAlibrary into a cell and preparing a culture of the obtained cell in thepresence of a lectin having an activity to inhibit the growth of saidcell; separating the grown cell from said culture and isolating fromsaid cell, a DNA which encodes α2→3 sialyltransferase; and introducingsaid DNA into a vector downstream the position of a promoter therein. 7.A process for production of an α2→3 sialyltransferase having an aminoacid sequence as designated by Seq. ID: 2 or 7, which comprises thesteps of:preparing a culture of a cell containing a recombinant vectoraccording to claim 3 or 4 in a medium and allowing α2→3sialyltransferase produced by said cell to accumulate in said culture;and isolating α2→3 sialyltransferase from said culture.
 8. A processaccording to claim 5, wherein said animal cell is a TYH cell or a humanmelanoma WM266-4 cell.
 9. A process according to claim 6, wherein saidanimal cell is a TYH cell or a human melanoma WM266-4 cell.
 10. Aprocess according to claim 5, wherein said lectin is Ricinus communis120 lectin.
 11. A process according to claim 6, wherein said lectin isRicinus communis 120 lectin.
 12. Plasmid pUCl19-LEC.
 13. PlasmidpUCl19-WM17.
 14. A cell containing a recombinant vector according toclaim 3 or
 4. 15. A method for adding sialic acid at non-reducingterminus in the lactosamine structure of a glycoprotein or a glycolipidin α2→3 linkage by use of a cell according to claim
 14. 16. A method forconverting non-reducing terminus of sugar chains on glycoproteins orglycolipids into sialyl-Le^(x) structure by use of a cell according toclaim
 14. 17. A method for detecting the expression of an α2→3sialyltransferase having an amino acid sequence as designated by Seq.ID: 2 or 7 by hybridization using a cDNA according to claim 1 or
 2. 18.A method for detecting the expression of an α2→3 sialyltransferasehaving an amino acid sequence as designated by Seq. ID: 2 or 7 bypolymerase chain reaction using synthesized oligonucleotides as probeswhich are designed based on the nucleotide sequence of cDNA according toclaim 1 or
 2. 19. A method for suppressing the expression of an α2→3sialyltransferase having an amino acid sequence as designated by Seq.ID: 2 or 7 by use of an oligonucleotide which contains part or all ofthe nucleotide sequence of a cDNA according to claim 1 or
 2. 20. AnEscherichia coli strain containing a recombinant vector according toclaim 3 or
 4. 21. Escherichia coli strain HB101/pUCl19-LEC (FERMBP-3625).
 22. Escherichia coli strain HB101/pUCl19-WM17 (FERM BP-4013).